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Immundiagnostik NewsletterIn Focus.......................................................................................................................... 125 years of successful immunoassay development for niche markets ............................................................................................ 1

Product News........................................................................................................... 2MRSA PCR for a fast & direct detection of Staphylococcus aureus in clinical samples .......................................................................................... 2Reliable routine marker of iron deficiency .............................................. 2Lactoferrin ELISAs complement IBD screening panel .............. ........ 2Special buffer ensures stability of calprotectin in stool samples .. 225(OH) Vitamin D Xpress ELISA for a quick routine analysis ........... 2

ID-Products in the Field........................................................................................... 3Serum OPG determination as survival rate predictor in prostate cancer patients with bone metastasis.............................. 3

Meeting Summaries................................................................................................ 3Vitamin D central topic in fall meetings ................................................. 3MEDICA - Forum for dialogue .................................................................... 3

Praxis to the Point...................................................................................................... 41-point calibration or standard curve? It depends on what you need! ...................................................................... 4

Events ............................................................................................................................. 4

02/2011

In Focus25 years of successful immunoassay development for niche marketsImmundiagnostik.celebrates.its.business.anniversary.and.continues.to.explore.new.opportunities

In 1986, Immundiagnostik was founded by Dr. Franz Paul Armbruster with the strategic goal to de-velop and produce immunoassays that fit the various needs of clinical routine and research. What started out as a 3-men company in a small flat quickly gained speed and since then developed into a flourishing global operating business with more than 60 employees.

The company's success is based on its unique strategy, which has changed little since the begin-nings: We monitor the latest scientific developments and select clinically relevant parameters and disease markers, that are useful for diagnosis, therapy monitoring or research. We then develop high quality test systems according to market needs. Over the years, our broad ELISA portfolio has thus been supplemented by HPLC, LC-MS/MS, PCR products and immunochemicals.

Immundiagnostik continues its progressive path by exploring new opportunities in different mar-kets around the world, by evaluating companion diagnostics options and by collaborating with research institutions.

Our achievements are the result of a dedicated team as well as of successful relations with our customers and business partners. We would like to use this opportunity to express our gratitude to all our stakeholders for cooperating with Immundiagnostik – some of you now for more than two decades. We look forward to working with you in the future!

The Immundiagnostik team wishes you Merry Christmas, peaceful holidays, and a healthy, successful New Year!

25Years

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weltweit

einzigartig!

PRODUCT NEWS

MRSA PCR for a fast & direct detection of Staphylococcus aureus in clinical samples

Methicillin-resistant Staphylo-coccus aureus (MRSA) is respon-sible for several difficult-to-treat infections in humans. The bacte-rium has developed resistance to beta-lactam antibiotics and cephalosporins and is especially troublesome in hospitals and nursing homes. The MutaPLEX® MRSA real time PCR kit is suitable for the direct detection of MRSA in clinical specimens, containing both coagulase-negative staphylococci and methicillin-resistant S. aureus. No pathogen cultivation necessary, detection in less than two hours.

MutaPLEX® MRSA, 96 tests (KG190396)

Reliable routine markers of iron defi ciency

Many routine markers of iron defi ciency (e.g. hemoglobin, transferrin, ferritin) are not ade-quate to discover pre-anemic stages and are infl uenced by infections or infl ammation. Zinc protoporphyrin is a sensitive and specifi c indicator of the iron status, independent of infections and in-fl ammation. Our zinc protoporphyrin HPLC kit al-lows the easy determination in whole blood in only 10 min. running time. In addition, our novel Hepcidin LC-MS/MS kit ena-bles the precise monitoring of iron homeostasis. Hepcidin is an early predictive marker of functio-nal iron defi ciency and allows the diff erentiation between Anemia of Chronic Disease (ACD) (high serum level) and Iron Defi ciency Anemia (IDA) (low serum level). Quantitative determination in serum, tuning kit available.

Zinc Protoporphyrin HPLC (KC2700)

Hepcidin LC-MS/MS (KM4000) Hepcidin Tuning Kit (KM 4001)

Special buff er ensures stability of calprotectin in stool samples

Our ELISAs for the deter-mination of calprotectin in stool samples contain a spe-cial extraction buff er which maintains the stability of the parameter for four weeks at room temperature. This ex-ceptional stability enables accurate analysis and prolonged storage of sample material. For a fast and economical calprotectin measurement we recommend our 1-hour assay – also available as 1-point calibration test.

PhiCal® Calprotectin ELISA, 1-pt calibration (K 6967)

PhiCal® Calprotectin ELISA (K 6927)

25(OH) Vitamin D Xpress ELISA for a quick routine analysis

The 25(OH) Vitamin D Xpress ELISA is the latest member of our broad product portfolio in vitamin D analysis and ac-complishes a convenient and precise status determination in only four hours. The automatable test measures 25(OH) vitamin D quan-titatively in serum, even in small sample volumes. The protocol does not require overnight incubation or cooling – the ideal assay for the status analysis of 25(OH) vitamin D in daily routine.

25(OH) Vitamin D Xpress ELISA (K 2107)

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MEETING SUMMARIES

Vitamin D central topic in fall meetings

The assessment of vitamin D has changed: While in recent years the hormone has been discussed mainly as a regulator of bone metabolism, new clinical data reveal the signifi cance of vitamin D in other organ systems. A lack can have severe con-sequences and has been associated with an eleva-ted risk for a variety of diseases (e.g. cardiovascular conditions, diabetes, cancer, low immunity).

The precise determination of vitamin D in routine and research has been a central business area at Immundiagnostik for years. Today, our comprehen-sive product portfolio covers the complete range of 25(OH) Vitamin D and 1,25(OH)

2 Vitamin D dia-

gnostics – from ELISA to LC-MS/MS. This unique competence has been presented by our fi eld force at relevant meetings, including the ASBMR in San-Diego (16.-20.09.), leading international congress for bone research, at the national vitamin D sym-posia in Freiburg (14.-15.10.) and Schwerin (09.11.) and at the 7. interdisciplinary bone and muscle fo-rum bone in Berlin (25.-26.11.).

MEDICA – forum for dialogue

Like in previous years, this year's MEDICA fi t the motto "B2B": There were continuous meetings at the booth with our international distributors and national business partners. In addition, our team could welcome many regular customers as well as new visitors at the booth. Our product presentations focussed this year on our four core business areas gastroenterology, cardiovascular diseases, bone metabolism, and oxidative stress alongside with our molecular biology portfolio. Aside from extensive dialogues and business apointments, our guests en-joyed the informal get-together at our bar with wine and snacks.

Serum OPG determination as survival rate predictor in prostate cancer patients with bone metastasis

The authors of the actual japanese study examined the diagnostic accuracy of serum osteoclastoge-nesis markers for the detection of bone metastasis and for the prediction of mortality in prostate can-cer patients. They used Immundiagnostik ELISA kits to measure the concentration of the parameters OPG and RANKL in the serum of 201 subjects (51 prostate cancer patients with, 101 without bone metastasis as well as 49 healthy individuals). The statistical analysis subsequently identifi ed inde-pendent predictors of bone metastasis and cor-related both markers with the incidence of bone metastasis and survival rate: Elevated OPG levels in serum proved to be a signifi cant predictor of bone metastasis and a reliable, independent prognostic factor for survival probability in patients with bone metastasis. The RANKL serum concentration on the other hand exhibited no diagnostic value.

The authors conclude from these data that OPG is an important parameter for predicting bone meta-stasis and that OPG could be an additional useful parameter for the assessment of the clinical classifi -cation of prostate cancer patients.

OPG ELISA (KB1011) total sRANKL ELISA (K1016)

ID-PRODUCTS IN THE FIELD

Kamiya N et al. (2011) „Signifi.cance.of.serum.osteopro-tegerin.(OPG).and.receptor.activator.of.nuclear.factor.κB.ligand.(RANKL).in.Japanese.prostate.cancer.patients.with.bone.metastasis“...Int J Clin Oncol 16(4):366-72

the bone metastases of patients with PCa [14, 15]. OPG,

also produced by osteoblasts and stromal cells, is a soluble

decoy receptor for RANKL, and its binding to RANKL

inhibits bone resorption and prevents bone loss by pre-

venting RANKL–RANK interactions [16]. In transgenic

animal models over-expressing OPG and in RANK knock-

out mice, osteopetrosis was observed due to lack of

osteoclasts. In these models, administration of RANKL

resulted in the development of osteoporosis associated with

hypercalcemia [21, 22]. RANKL is a member of the TNF

superfamily that is expressed by osteoblasts, bone marrow

stromal cells, and activated T-cells [23, 24]. RANKL

stimulates bone resorption by binding to its receptor,

RANK, on the surface of osteoclasts and their precursors,

thereby promoting osteoclast formation, function, and

survival [25, 26]. Therefore, the RANKL/RANK/OPG

system represents a key regulatory mechanism in

osteoclastogenesis.

Reverse transcription-polymerase chain reaction and

immunohistochemistry have been used frequently to

investigate the expression of RANKL, RANK, and OPG.

Brown et al. [15] reported that PCa with bone metastases

were consistently immunoreactive for both OPG and

RANKL compared with non-osseous metastases or primary

PCa. Similarly, Chen et al. [27] found that surgical biopsy

specimens of metastatic PCa had significantly higher

expression of RANKL, RANK, and OPG than those of

non-metastatic PCa.

Serum markers can be determined frequently and easily,

with negligible disturbance to the patient. Several clinical

studies have assessed the diagnostic efficacy of osteo-

clastogenesis markers for the detection of bone metastases

in PCa. To our knowledge, however, we report the first

combined serum measurements of RANKL and OPG in

Japanese patients with PCa. In a study of German men,

Jung et al. [28] measured serum OPG and RANKL in PCa

patients, in presumably healthy men, and in patients with

benign prostatic hyperplasia (BPH). They reported that

serum OPG levels were increased in patients with bone

metastases, while those with localized cancer or lymph

node metastases had values similar to those in presumably

healthy controls and patients with BPH. Serum RANKL

levels did not differ among the control, BPH, and PCa

subgroups. Furthermore, they commented that ROC anal-

ysis data indicated that OPG had better diagnostic accuracy

than serum bone turnover markers for detecting bone

metastases in PCa cases. Our present study supports their

data. We found that serum OPG levels were significantly

elevated in patients with bone metastasis and significantly

predicted bone metastasis on multivariate stepwise logistic

regression analysis. Our ROC analyses also showed that

serum OPG level was the most reliable predictor of bone

metastasis. Jung et al. [28] found that serum RANKL

levels were not significantly increased in PCa patients with

bone metastasis compared with those with BPD and PCa

without bone metastasis. Thus, they commented that the

Table 4 Cox proportional hazards analysis of factors predicting time to cause-specific death in PCa patients with bone metastasis

Univariate analysis Multivariate analysis

Relative HR 95%CI p value Relative HR 95%CI p value

Gleason score (\8/C8) 4.341 0.984–19.153 N.S.

EOD score (1–3/4) 13.515 2.203–82.912 0.0049 12.351 1.882–81.048 0.0088

T stage (3/4) 0.864 0.300–2.489 N.S.

N stage (N0/N1) 2.204 0.502–9.684 N.S.

PSA (\3523.1/C3523.1) 0.504 0.162–1.568 N.S.

OPG (\181.1/C181.1) 3.111 1.007–9.608 0.0458 3.670 1.142–11.796 0.0290

RANKL (\363.9/C363.9) 0.438 0.058–3.308 N.S.

Hb (\11.0/C11.0) 0.281 0.095–0.831 0.0218 0.528 0.139–2.001 N.S.

Ca (\9.4/C9.4) 1.230 0.146–10.341 N.S.

CI confidence interval, HR hazard ratio, EOD extend of disease on bone scan, PSA prostate-specific antigen, OPG osteoprotegerin, RANKLreceptor activator of nuclear factor jB ligand, SRE skeletal-related event, Hb hemoglobin, Ca calcium, N.S. not significant

Fig. 1 Cause-specific survival curves in M1b prostate cancer patients

with serum osteoprotegerin (OPG)

Int J Clin Oncol

123

Fig.: Cause-specifi c survival rate in prostate cancer pati-ents (M1b) correlates with the serum OPG concentration.(Fig. 1 from Kamiya et al., 2011)

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Praxis to the Point

1-Point calibration or standard curve? It depends on what you need!

The majority of our ELISA-Tests is available in two versions: As regular kit with fi ve standards for the calibration of a standard curve or as 1-point calibration assay with only one calibrator. Since both tests are comparable in price and since both are CE-certifi ed and automatable the quesion arises:

Which kit is the right one for your routine?

In our experience, this decision depends on the special needs of your laboratory. The 1-point calibration operates with a signifi cantly reduced number of calibrators which have to be determined in each run. Therefore, more samp-les can be measured per kit. This procedure is economical, especially for a medium amount of samples.

Some customers hesitate to buy a 1-point calibration assay because they mistrust the accuracy of the data – a worry without reason: The 1-point calibration of a test is based on a statistically signifi cant amount of runs which determi-ne the standard curve. The resulting function of the curve then defi nes the calibration of the 1-point calibration test. All the customer needs to do for result evaluation is to use a four parametric logit-log model based on the standard curve of the respective kit lot and the calibrator value. All essential standard curve information is provided on the QC data sheet (see example on the fi gure to the right). In ad-dition, a blank has to be measured to determine the back-ground absorption. The fi xation of the calibration curve by the two data points blank and calibrator equalize possible data deviations.

The statistic validation of the 1-point calibration assay thus warrants the same accuracy as the standard calibration.

If you are hesitant which calibration method to use our technical support team is happy to provide advice in all aspects.

tech.support@immundiagnostik.com

Immundiagnostik AGStubenwald-Allee 8a

D-64625 Bensheim Tel.: +49 (0) 62 51/70 19 00Fax: +49 (0) 62 51/84 94 30

info@immundiagnostik.comwww.immundiagnostik.com

02/2011

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Fig.:

Data sheet of the 1-point calibration version of the hemoglobin ELISA.

The. function. of. the. standard. curve. is.provided.and.has.to.be.adjusted.for.the.respective. run. by. the. OD-values. of. the.calibrator.and.the.blank.

EVENTS

• ARAB HEALTH23. - 26. January 2012, Dubai / United Arab Emirates Booth No. RF38

• ECCO (European Crohn's & Colitis Organisation) Congress16. - 18. February 2012, Barcelona / SpainBooth No. 17