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DIARECT Newsletter No. 2/2006
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Dear Madame or Sir, |
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November is Medica time and in anticipation of the upcoming fair in Duesseldorf (November 15-18, 2006), we would like to invite you to meet with us to discuss new antigens, current projects and the many prospects for the future. The DIARECT management will be attending Medica Wednesday and Thursday (15 & 16/11/05) all day, and we are looking forward to seeing you there. To arrange an appointment, please, give us a call or send an email.
Pleasant reading!
Your DIARECT team
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dsDNA antibodies |
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For nearly half a century dsDNA autoantibodies have been recognized as a main serological parameter in the in-vitro diagnosis of systemic lupus erythematosus (SLE). However, the answer to the question regarding the clinically most useful method will probably require another 50 years. Regardless of the method you may have chosen - Crithidia luciliae IIFT, FARR type immunoradiometric assay or ELISA - there is always the need for a reliable and reproducible calibrator and standard serum/autoantibody.
In an earlier DIARECT newsletter we introduced a pair of human monoclonal autoantibodies to dsDNA that ideally fulfil these prerequisites. Here we would like to present data generated using these monoclonal autoantibodies in three different assay systems. Whereas the reactivity of one of these monoclonal antibodies (mAb33) toward dsDNA (also nucleosomes as well as, interestingly enough, histones) correlates almost perfectly to the International Standard Serum against dsDNA (Wo80) in an ELISA (Fig. 1), the other (mAb32) can easily be used to calibrate FARR-type immunoprecipitation assay (Fig. 2) and, moreover, can be employed in immunofluorescent assays with Crithidia luciliae (Fig. 3).
The subtypes of the monoclonal antibodies are IgG1 (mAb33) and IgG3 (mAb32), which means that both antibodies can easily be detected by standard anti-human conjugates.
We would like to thank Prof. R.-L. Humbel (Luxembourg) and J. Westermann (Hamburg, Germany) for cordially providing their data.
| 12300 | dsDNA (plasmid) | 0.1 mg | | 12301 | | 1.0 mg | | 33200dsDNA-mAb32 (suitable for FARR, Crithidia) | trial sample | | 33201 | | bulk size | | 33300 | dsDNA-mAb33 (suitable for ELISA) | trial sample | | 33301 | | standard size |
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M2 - mitochondrial antibodies in PBC patients |
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Primary biliary cirrhosis (PBC) is a severe autoimmune liver disease accompanied by destruction of intrahepatic bile ducts.
Antimitochondrial autoantibodies (AMA) are a characteristic serological feature in PBC patients and were first described in 1965, but it was not until 1988 when two groups independently identified members of 2-oxo acid dehydrogenase complex (2-OADC) as major molecular targets for this type of autoantibodies (for review see 1). These include the E2 subunits (dihydrolipoamide transferases) of pyruvate dehydrogenase complex (PDC-E2), the E2 subunit of branched chain 2-BCOADC (BCOADC-E2), the E2 subunit of 2-oxoglutarate dehydrogenase complex (OGDC-E2) and the dihydrolipoamide dehydrogenase binding protein (E3BP). These target antigens are located in the mitochondrial matrix, associated with the inner membrane, and catalyze the oxidative decarboxylation of various alpha-keto-acid substrates (1).
The most common reactivity of AMA positive PBC sera is against PDC-E2. Whereas some patients have AMA that react with PDC-E2 alone (95%), most patients also show reactivity against OGDC-E2 (39-88%) and/or BCOADC-E2 (53-55%). This was exploited in 1996 when Moteki et al. reported on an engineered fusion protein comprised of the lipoyl domains of the three different proteins. When this fusion protein was employed in an ELISA, they were able to detect AMA's in a very sensitive and specific manner (2).
We would like to introduce our M2 antigen, which is now ready for evaluation. It represents a technically rather plain approach insofar as it is composed of full-length PDC-E2, BCOADC-E2 and OGDC-E2 that were first individually tested. We are greatly indebted to Prof. R.-L. Humbel (Luxembourg) for his enthusiasm and willingness to critically evaluate these M2 antigen single components with numerous clinically defined PBC patient sera (Fig. 2). Since one component alone is clearly not sufficient for unequivocally determining AMAs, we determined that a blend of these proteins would represent the optimal antigen (Fig. 1)
Please contact us for more information or for your free sample.
Ref. (1) Gershwin, M. E. (2000) Primary biliary cirrhosis: an orchestated immune response against epithelial cells. Immunol. Rev. 174, 210-25
Ref. (2) Moteki, S. (1996) Use of a designer triple expression hybrid clone for three different lipoyl domains for the detection of antimitochondrial autoantibodies. Hepatology 24, 97-103
| 18000 | M2-Antigen | 0.1 mg | | 18001 | | 1.0 mg | |
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DIARECT antigen reservation system |
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We would like to invite all our customers to take advantage of the proven DIARECT antigen reservation system. What does that mean for you?
We guarantee the reservation of a specified amount of antigen(s) (up to a whole production run). Usually the amount of antigen under reservation is sufficient for our customers' manufacturing needs for at the most two years. You simply inform us of your antigen requirements for this period, and after approximately 6 months we will give you feedback regarding the accuracy of your estimate. The reservation system also includes providing the customer early enough with samples of new lots of the respective antigen. This allows for a smooth transition when evaluating for the qualification of a new lot for subsequent reservation.
For you there are two clear advantages:
* Security of supply with no additional costs
* No need for continual readjustment and fine tuning of your assay because of new antigen lots.
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HRP Conjugate Stabilizers: Competitor Evaluation |
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A study was performed to evaluate the efficacy of SurModics' StabilZyme® HRP Conjugate Stabilizer in comparison to other commercially-available HRP conjugate stabilizers using an accelerated stability format. Results showed that SurModics' StabilZyme HRP Conjugate Stabilizer was superior to the other eight stabilizers tested after 9 months at 37ºC by retaining >60% activity. This retained activity at 37ºC is equivalent to >5 years stability if stored at 4ºC.
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The following test procedure was utilized for this study:
1. An anti-rabbit IgG peroxidase conjugate was diluted 1:10.000 in each HRP conjugate stabilizer. Individual 4ºC and 37ºC samples of each stabilizer were prepared for each time point to be tested (24 hours, 7 days, 1 month, 3 months, 6 months and 9 months).
2. At each time point, one 4ºC sample and one 37ºC sample were analyzed using the following standard ELISA procedure:
a. Aliquots of 100 µL per well of the coating solution (1:5.000 dilution of an anti-gliadin IgG) were added to corresponding wells of microtiter plates. The plates were incubated at 37ºC for 1 hour and then washed.
b. Aliquots of 150 µL of a blocking/stabilizing solution (e.g. StabilCoat Immunoassay Stabilizer) were added to corresponding wells of the microtiter plates which were then incubated at room temperature for 30 minutes and then washed.
c. Aliquots of 100 µL of the anti-rabbit IgG-HRP conjugate-containing stabilizers were added to the corresponding wells of microtiter plates which were then incubated at 37ºC for 1 hour and then washed.
d. Aliquots of 100 µL per well of a prepared chromagen solution were added to the microwells, and the plates were incubated for 15 minutes at room temperature. Color development was stopped with the addition of 50 µL per well of 1N H2 SO4.
3. A commercial plate reader was used to determine the OD values in each of the microtiter plate wells. The percent retained activity was calculated by comparing the OD values of the product samples at 4ºC and 37ºC: % Retained Activity = Net OD37ºC ÷ Net OD4ºC x 100
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