Figure 1.Biotinylated Bio-marker
In many ways, a conjugated bio-marker can be the solution to different problems arisen in a new IVD test development. A common problem caused by most of the surfaces is protein denaturation due to the relatively high surface by hydrophobicity. Additionally, there may be larger steric influence on binding events due to the close proximity of the surface and the sensor molecules. The specific orientation may also improve the stability of attached proteins and increase the sensitivity of the assay by exposure of its antigenic regions.
Conventional conjugation methods generally work well with antibodies; however, results with many antigens of less well-established structures are less constant. This is probably the main reason why double-antigen sandwich ELISAs (DAS-ELISA) are not as commonly used for antibody detection as indirect ELISA.
To circumvent the effect on antigenic structure caused by conventional chemical conjugation that could result in decrease in sensitivity of DAS-ELISAs, Rekom Biotech has developed a product line of biotinylated antigens, offering to our customers the possibility to buy their favourite antigen but conjugated in its C-terminus. This molecule allows the specific interaction of the biotinylated antigens to the streptavidin protein.
The extremely specific and high affinity binding of biotin by avidin and/or streptavidin (Kd ≈ 10-14M) results in specific detection systems of very high sensitivity. A clear advantage of this system is that with a common strep-HRP, we can obtain conjugated complex of all our references without the necessity of performing the peroxidation of each reference.
The biotin is fused to a linker which maintains the molecule away to the antigen surface, avoiding steric hindrance between the biotin and the antigenic regions involved in Ab-binding. Thus, the Ab interaction will not be compromised with the conjugation.
Figure 2. Interaction between an antigen (green) vs Fab region of an Ab (pink). In red are specified the amino acids that usually are used in conjugation procedures. Some of these amino acids are part of the epitope region of the antigen. The conjugation would interfere with the Ab-binding, decreasing the antigenic capacity of the bio-marker.
As there is just one biotin per protein molecule, our conjugated bio-markers will show a higher inter-lot reproducibility and this will facilitate the reproducibility of our customers´ IVD test also.
- Antigen orientation in streptavidin-coated plates.
- Detection in IgM capture assays.
- Detection in DAS assays.
- Bonding to gold and other nanoparticles coated with streptavidin.
Rekom Biotech biotinylated bio-markers
||Chimeric recombinant antigen which contains several antigenic determinants for CMV
||DNA polymerase processivity subunit
||Viral capsid antigen
||Parasite kinesin-related antigen
||Recombinant chimeric antigen for Treponema pallidum
|Timothy grass pollen (Phleum pratense)
||Phl p 5a
|Wheat (Triticum aestivum)
||Tri a 19
||Omega-5 gliadin, seed storage protein
Human cytomegalovirus (HCMV) is endemic to populations throughout the world, and 50-80% of the adults in developed nations are sero-positive for this virus. Commercial ELISAs for detecting IgG are very sensitive and specific. Nevertheless, there are several problems regarding IgM detection. One of the difficulties that the diagnostic laboratory has faced over the past 10 years is the lack of agreement between commercial tests for the detection of CMV-specific IgM. This lack of agreement has its roots in the different viral preparations used to detect IgM antibodies to CMV. Rekom Biotech has performed the design of a new CMV recombinant polyepitope chimeric antigen (RAG0109), composed by different sensitive and specific antigenic determinants from some CMV proteins. This recombinant polyepitope chimeric antigen was tested in parallel with other CMV bio-markers such as the Rekom Biotech CMV recombinant proteins pp150 (RAG0091) and pp52 (RAG0090) and pp65 (RAG0016). The in house indirect IgM ELISA assay was carried out by using a battery of pre-validated sera with the commercial ELISA capture IgM test from Vidas:
Figure 1: Indirect IgM ELISA assay. Proteins were coating the plates at a final concentration of 1 µg/ml. All these experiments used anti-IgG as sorbent and anti-IgM-HRP. Sera were used in a 1:100 dilution. P means pre-validated sera by the Vidas test as positive; N means pre-validated sera by the Vidas test as negative.
These preliminary experiments suggest that the recombinant polyepitope chimeric antigen designed and produced by Rekom Biotech, RAG0109, recovers some pre-validated IgM positive sera which are not initially detected by two of the best antigens described in bibliography for CMV diagnosis: pp52 and pp150, showing the presence in merely one antigen of some important epitopes for this specific diagnostic.
To find more interesting information on regards recombinant antigen RAG0109, please click here.
Recently, ReKom Biotech has published a white paper concerning good practices for antigen titration. This may be seen as an old issue. However, from the point of view of a data analyst the approaches in the literature are not really convincing. Titration experiments should be designed to obtain the most information on how to use the antigen at the lowest cost, that is, with the lowest number of experiments. It should be noted that, while the antigen may be used for quantitative or qualitative analysis, the titration experiments should be designed differently in both cases. The published paper is focused on qualitative analysis since the main goal of the titration experiments in ReKom Biotech is to convince our customers of the high quality of our products. We have devoted considerable efforts to develop titration procedures to indicate the customers at which concentration the antigen best discriminates between positive and negative serum of a given disease. Existent and widely used procedures present several drawbacks. For instance, in most standard titration experiments, such as in Chequerboard Titration (CBT), one single positive and negative sera are employed. From a statistical point of view, this is not a good approach since results are very dependent on the specific sera chosen. On the other hand, when several positive and negative sera are included in the experiment, the results are not assessed with the adequate tools. For instance, we could not find a single paper which compared the outcomes of positive and negative sera with a simple test of significance. The white paper we have published is intended to show clients that we use well-grounded methods to measure the quality of our products, and also to give details on these methods.
The possibility to use ammonium hydroxide to control the pH in fed-batch microbial fermentations would have the advantage over sodium hydroxide that it could be used as a nutrient thus avoiding the increase of the media osmolality. However, this reaction equilibrium should be analysed carefully:
NH3 + H2O = NH4+ + OH-
At 25ºC and infinite dilution:
[NH4+][ OH- ]/[ NH3] = 0.000018
And when temperature or pH rises, free ammonia could be given off, polluting the outlet gas of the bioreactor with this extremely toxic component.
NH4+ + OH- = NH3 + H2O
Due to this reason, ammonium hydroxide only could be used as pH corrector in solutions which are slightly acid, thus in the case of pH = 5, where pOH = 14-5 = 9.
[ NH4+](0.000000001)/[ NH3] = 0.000018
[NH4+]/[ NH3] =18000
In this particular case, the main quantity of the ammonium hydroxide appears to be in the form of ammonium ion which will be consume as nitrogen source and therefore it could be used to rise the pH without the production of free ammonia.
For this reason, ammonium hydroxide shouldn´t be used to adjust the pH in fed-batch microbial fermentations of Escherichia coli where pH 7 is necessary, but it would be very convenient in the case of the methylotrophic yeast Pichia pastoris which optimal growth takes places at pH 5.
In fed-batch microbial fermentations, O2 is not only important as a nutrient, but also it has important effects on metabolism and physiology. There are a number of potential oxygen-sensitive steps which could affect recombinant expression: (i) the results of low oxygen, (ii) the results of high oxygen and (iii) the results of oxygen shifts.
- A shift to anaerobic metabolism can produce accumulation of by-products such as acetate and ethanol which have a negative impact on process performance. For example, acetate can limit productivity of recombinant E. coli MC1016 harbouring the plasmid for hGH production (Jensen and Carlsen, 1990). On the contrary, this partial anaerobiosis, often referred as “microaerobic growth” sometimes is beneficial for expression of certain heterologous genes in E. coli (Tseng et al., 1996). There are several effects under anoxic conditions:
- Regulation of amino acid synthesis by oxygen
- Effects in amino acids depletion
- Plasmid replication
- Hemoglobin expression to improve productivity
- Exposure to elevated oxygen partial pressures (due to OTR limitation or in large bioreactors) can lead to: (Konz et al, 1998).
- An oxidative damage to proteins in five different classes: metal-catalyzed oxidation, disulfide formation, methionine sulfoxide formation, oxidation of iron-sulfur centers and glycation and PUFA conjugation
- Degradation of oxidized proteins
- DNA oxidation and mutation
- Oxidation of free aminoacids
- Oxygen fluctuations can be also detrimental to protein expression. If we shift to oxygen-enriched air during expression of our heterologous gene, our product quality may initially decrease due to the oxidative conditions and afterwards the quality may increase to a new state as tress regulons are induced.
To conclude: fluctuating DO in fed-batch microbial fermentations is not a trivial process which can affect not only the amplification of plasmids in E. coli, but also the quality of our final recombinant protein, sometimes with dramatic consequences.
- Jensen, E. B and S. Carlsen. 1990. Biotechnol. Bioeng. 36: 1-11.
- Tseng, C. P., J. Albretch, and L. P. Gunsalus. 1996. J. of Bacteriol. 178: 1094-1098.
- Konz, J. O., J. King, and C. L. Cooney. 1998. Biotechnol. Prog. 14: 393-409.