SERION ELISA classic Legionella pneumophila 1-7 IgG/IgM

CONTENTS

1. INTENDED USE: For sale in the U.S. for Research Use Only. Not for use in diagnostic
procedures.

2. BACKGROUND

3. SERION ELISA classic - TEST PRINCIPLE

4. COMPONENTS OF THE KIT

5. MATERIAL REQUIRED BUT NOT SUPPLIED

6. STORAGE AND STABILITY

7. TEST PROCEDURE SERION ELISA classic

7.1 Evidence of deterioration

7.2 Sample preparation and storage
7.3 Preparation of kit reagents
7.4 Overview - test procedure
7.5 Test procedure

8. TEST EVALUATION

8.1 Single-point quantification with the 4PL method

8.2 Criteria of validity
8.3 Calculation SERION ELISA classic
Legionella pneumophila 1-7 IgG/IgM (quantitative)

9. STATEMENTS OF WARNING

10.1 Statements of warning

10.2 Disposal

10. BIBLIOGRAPHY

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 SERION ELISA classic Legionella pneumophila 1-7 IgG/IgM

Enzyme Immunoassay for detection of human antibodies (IgG/IgM)

For sale in the U.S. for Research Use Only. Not for use in diagnostic
procedures.

IgG-Kit (quantitative) order number: ESR106G

IgM-Kit (quantitative) order number: ESR106M

Tests evaluated : Dade Behring BEP ® III / BEP ® 2000, DSX, manually

1. INTENDED USE

SERION ELISA classic Legionella pneumophila 1-7 IgG/IgM are quantitative or

qualitative tests for detection of human antibodies in serum or plasma against Legionella
pneumophila 1-7. For sale in the U.S. for Research Use Only. Not for use in diagnostic
procedures.

2. BACKGROUND
Legionella belongs to the species of legionellaceae, genus Legionella. It is a gram negative,
aerobic, pleomorphic, non-spore forming bacillus which is motile by means of a single or
multiple polar or subpolar flagella. Currently, the genus includes 39 Legionella species.
Immunologic diversity within species is reflected in the creation of serogroups. The most
important human pathogen is the species Legionella pneumophila which comprises 14
serologic groups.

Legionella are ubiquitous bacteria which reside in surface and drinking water and multiply
in amoebae and other protozoa. Warm water systems with a temperature of 25-45°C are
ideal for multiplication. After transmission to humans in aerosols, the bacteria are
phagocytized by alveolar macrophages. So called evasion mechanisms enable bacteria not
only to survive but also to multiply within a phagocytic cell.

Due to non-specific clinical symptoms, diagnosis is based on laboratory techniques such as

direct pathogen detection, derived antigen detection, or detection of specific antibodies.

Direct pathogen detection in culture or in direct immunofluorescence assays (DFA) are of

outstanding importance because fast diagnosis is often decisive for the patient. Sputum,
tracheobronchial secretion, and pleura punctates are ideal samples. However, sensitivities
of only 50-60 % are described for both techniques.

To compensate for the above mentioned drawbacks of direct detection methods, serologic
test systems are widely used. Since multiple serogroups of L. pneumophila are human
pathogens, pool antigens are favored. In the SERION ELISA classic IgG/IgM, a mixture of
serogroups 1-7 is bound to the solid phase.

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3. SERION ELISA classic - TEST PRINCIPLE

Microtest plates are coated with antigens. This constitutes the solid phase. Sample is
added to the plates and any antibodies specific for the antigen present will bind to the solid
phase. After removal of unbound material, anti-human IgG, IgA or IgM conjugated to an
enzyme (alkaline phosphatase) is allowed to react with the immune complex. After
removal of excess conjugate by washing, an appropriate substrate (para-
nitrophenylphosphate) is added, with which the conjugated enzyme reacts producing a
colored derivative of the substrate. The color intensity is proportional to the level of
specific antibody bound and can be quantified photometrically.

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4. COMPONENTS OF THE KIT

Test components amount /

volume
Break apart microtiter test strips each with 8 antigen coated single wells (altogether 96), 12
1 frame
the coating material is inactivated

Standard serum (ready-to-use) 2 x 2 ml

Human serum in phosphate buffer with protein; negative for anti-HIV-Ab, anti-HBs-Ag
(Hepatitis B-Virus-surface antigen) and anti-HCV-Ab; preservative: < 0.1 % sodium azide
coloring: Amaranth O

Negative control serum (ready-to-use) 2 ml

Human serum in phosphate buffer with protein; negative for anti-HIV, anti-HBs (Hepatitis B-
Virus-surface antigen) and anti-HCV; preservative: < 0.1 % sodium azide
coloring: Lissamin green V

Anti-human-IgG-, IgA-, IgM-conjugate (ready-to-use) 13 ml

Anti-human-IgG, -IgA, -IgM from goat (polyclonal), conjugated to alkaline phosphatase,
stabilized with protein stabilization solution
preservative: 0.01 % methylisothiazolone, 0.01 % bromnitrodioxane

Washing solution concentrate (sufficient for 1 litre) 33.3 ml

Sodium chloride solution with Tween 20, 30 mM Tris
preservative: < 0.1 % sodium azide

Dilution buffer 2 x 50 ml
Phosphate buffer with protein and Tween 20;
preservative: < 0.1 % sodium azide
0.01 g/l Bromphenol blue sodium salt

Stopping solution 15 ml
1.2 N sodium hydroxide

Substrate (ready-to-use) 13 ml
Para-nitrophenylphosphate, solvent free buffer
preservative: < 0.1 % sodium azide
(Substrate in unopened bottle may have a slightly yellow color. This does not reduce the
quality of the product!)

Quality control certificate with standard curve and evaluation table 1

(quantification of antibodies in IU/ml or U/ml)

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5. MATERIAL REQUIRED BUT NOT SUPPLIED

- common laboratory equipment

- for the IgM-ELISA: SERION Rf-Absorbent (Order no. Z200/20ml)
- photometer for microtiter plates with filter, wavelength 405 nm, recommended
reference wavelength 620 nm - 690 nm (e.g. 650 nm)
- incubator 37°C
- moist chamber
- distilled water

6. STORAGE AND STABILITY

Reagent Storage Stability

microtiter strips after opening at 2-8°C in closed aluminum bag with 4 weeks
(antigen) desiccant

Strips which are not used must be stored in the press-seal bag
of aluminum compound foil under dry and airtight
conditions!
control sera / after opening at 2-8°C until expiry date;
standard sera 24 months from
date of production

conjugate ready-to-use solution, at 2-8°C until expiry date

28 months from
Avoid contamination (sterile tips!) date of production

dilution buffer after opening at 2-8°C 24 months

Discard cloudy solutions!
until expiry date;
unopened 36 months from
date of production
washing solution concentrate after opening at 2-8°C until expiry date
working dilution at 2-8°C 2 weeks
working dilution at room temperature 1 week

Bottles used for the working dilution should be cleaned

regularly, discard cloudy solutions.

substrate ready-to-use solution at 2-8°C, protected from light! until expiry date
24 months from
Avoid contamination (sterile tips!) Discard when solution date of production
turns yellow (extinction against distilled water > 0.25).

stopping solution after opening at room temperature until expiry date

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7. TEST PROCEDURE SERION ELISA classic

7.1 Evidence of deterioration

Only use SERION ELISA classic reagents for test procedure, since all reagents are matched.
In particular standard and control sera are defined exclusively for the test kit to be used. Do
not use them in other lots. Dilution buffer, washing solution and substrate solution can be
used for all SERION ELISA classic kits irrespective of the lot and the test.

There are three different conjugate concentrations for each immunoglobulin class: LOW,
MEDIUM, HIGH

The classification is written on each label as follows:

e.g. IgG + lowly concentrated IgG conjugate

IgG ++ medium concentrated IgG conjugate
IgG +++ highly concentrated IgG conjugate

In rare cases the use of special conjugate is necessary to guarantee consistent quality for our
products. Special conjugates are produced in a separate lot and do not wear the “+” sign.
Therefore, special conjugates are not exchangeable with other conjugates.

Please pay close attention to notifications on labels!

Unopened, all components of the SERION ELISA classic kits may be used up to the dates
given on the labels, if stored at +2°C to +8°C. Complete stability and storage data are
described under 6. “Storage and Stability”..

Each reagent has been calibrated and optimized for the test. Dilution or alteration of these
reagents may result in a loss of sensitivity.

Avoid exposure of reagents to strong light during storage and incubation. Reagents must
be tightly closed to avoid evaporation and contamination with microorganisms since
incorrect test results could occur due to interference from proteolytic enzymes.

To open the press-seal bag please cut off the top of the marked side, only. Do not use the
strips if the aluminum bag is damaged or if the press-seal bag with remaining strips and
desiccant was not properly closed.

Bring all reagents to room temperature before testing.

Use aseptic techniques for removing aliquots from the reagent tubes to avoid
contamination. To avoid false positive results ensure not to contact or sprinkle the top-walls
of wells while pipetting conjugate. Be careful not to mix the caps of the bottles and/or vials.
Reproducibility depends on thorough mixing of the reagents. Shake the flasks containing
control sera before use and also all samples after dilution (e.g. by using a monomixer).

Be sure to pipette carefully and comply with the given incubation times and temperatures.
Significant time differences between pipetting the first and last well of the microtiter plate
when filling samples/control sera, conjugate or substrate may result in different
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„pre incubation“ times, which may influence the precision and reproducibility of the
results.

Optimum results can only be achieved if SERION ELISA classic instructions are followed
strictly.

The test is not valid, if the lot-specific validation criteria on the quality control certificate are
not fulfilled.

Inadequate washing will affect the test results:

The washing procedure should be carried out carefully. If the washing procedure is carried
out automatically follow the instruction manual of the respective washer. Flat bottom wells
are used for SERION ELISA classic. All wells should be filled with equal volumes of
washing buffer. At the end of the procedure ensure that the wells are free of all washing
buffer by tapping the inverted microtest plate on a paper towel. Avoid foam! Do not scratch
coated wells during washing and aspiration. If using an automated washer, ensure it is
operating correctly.

7.2 Sample preparation and storage

Lipaemic, hemolytic or icteric samples should only be tested with reservations although in
our testing no negative influence has been found. Obviously contaminated samples (serum
or plasma) should not be tested due to the risk of wrong results.

Serum or Plasma (EDTA, citrate, heparin) collected according to standard laboratory

methods are suitable samples.

Samples must not be thermally inactivated.

7.2.1 Sample preparation

Before running the test, samples must be diluted in dilution buffer (V1 + V2) as follows:

SERION ELISA classic Legionella pneumophila 1-7 IgG

V1 + V2 = 1+100 add 10 µl sample

each to 1000 µl dilution buffer

After dilution and before pipetting into the microtiter plate the samples must be mixed
thoroughly to prepare a homogenous solution.

SERION ELISA classic Legionella pneumophila 1-7 IgM

Rheumatoid factors are autoantibodies mainly of the IgM-class, which preferably bind to
IgG-immune-complexes. The presence of non-specific IgM-antibodies (rheumatoid factors)
can lead to false-positive results in the IgM-assay. Furthermore, the possibility exists, that

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weak-binding pathogen-specific IgM-antibodies are displaced by stronger-binding
IgG-antibodies. In this case, IgM-detection can lead to false-negative results. Therefore it is
necessary to pretreat samples with rheumatoid factor-absorbens prior to IgM detection
(SERION Rheumatoid Factor-Absorbent, Order-No. Z200 (20 ml/100 tests)).

Before running the test, rheumatoid factor-absorbent (V1) must be diluted 1+4 in dilution
buffer (V2).

V1 + V2 = 1 + 4 add 200 µl Rf-absorbent

each to 800 µl dilution buffer

Samples (V4) must be diluted in this Rf-dilution buffer (V3)

V4 + V2 = 1+100 add 10 µl sample

each to 1000 µl Rf-dilution buffer

7.2.2 Sample storage

The stoppered samples can be stored in a refrigerator up to 7 days at 2-8°C. Extended

storage is possible at ≤ -20°C.

Avoid repeated freezing and thawing of samples.

Diluted samples can be stored at 2-8°C for one week.

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7.3 Preparation of kit reagents

7.3.1 Microtest strips

Microtest strips in frame are packed with desiccant in an aluminum bag. Take
unrequired cavities out of the frame and put them back into the press-seal bag. Close
press-seal bag carefully to ensure airtight conditions.

7.3.2 Control sera / standard sera

Control and standard sera are ready-to-use and must not be diluted any further. They
can be used directly for the test run.

For each test run and for each test system - independent of the number of microtest
strips to be used - control and standard sera must be included. The cut-off-control
should be set up in duplicate. With the quantitative tests the standard serum should
also be set up in duplicate.

Do not treat control sera with Rf-absorbent.

7.3.3 Anti-human-IgG-, IgM- or IgA-AP-conjugate (ready-to-use)

Please do not mix up conjugates from different kits. They are optimized for each lot.
Conjugates are exchangeable as described in 7.1.
Avoid contamination of ready-to-use conjugates (please pour sufficient for test into a
secondary container to avoid repeatedly pipetting from the original bottle).

7.3.4 Washing solution

Dilute washing buffer concentrate (V1) 1:30 with distilled water to a final volume of
V2 .

Example:
buffer concentrate (V1) final volume (V2)
33.3 ml 1000 ml
1 ml 30 ml

7.3.5 Dilution buffer for samples (ready-to-use)

7.3.6 Substrate (ready-to-use)

To avoid contamination use gloves. For pipetting substrate solution use sterile tips
only!

7.3.7 Stopping solution (ready-to-use)

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7.4 Overview - test procedure

Legionella pneumophila 1-7

IgG/IgM quantitative

in case of IgM-detection absorption of rheumatoid factor!

sample dilution

1 + 100
Ø

Pipette diluted samples and ready-to-use control sera /

standard sera into the microtest wells (100 µl)
Ø

INCUBATION 60 min./37°C
moist chamber
Ø

WASH
Ø

Pipette conjugate solution (100 µl)

Ø

INCUBATION 30 min./37°C
moist chamber
Ø

WASH
Ø

Pipette substrate solution (100 µl)

Ø

INCUBATION 30 min./37°C
moist chamber
Ø

Pipette stopping solution (100 µl)

Ø

READ EXTINCTION AT 405 nm

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7.5 Test procedure

1. Place the required number of cavities in the frame and prepare a protocol sheet.

2. Add each 100 µl of diluted sample or ready-to-use controls into the appropriate wells
of microtest strips. Spare one well for substrate blank, e.g.:
IgG/IgM quantitative

well A1 substrate blank

well B1 negative control
well C1 standard serum
well D1 standard serum
well E1 sample 1....

3. Sample incubation for 60 minutes (+/- 5 min) at 37°C (+/- 1°C) in moist chamber

4. After incubation wash all wells with washing solution (by automated washer or
manually):
- aspirate or shake out the incubation solution
- fill each well with 300 µl washing solution
- aspirate or shake out the washing buffer
- repeat the washing procedure 3 times (altogether 4 times!)
- dry by tapping the microtest plate on a paper towel

5. Addition of conjugate
Add 100 µl of IgG/IgM-conjugate (ready-to-use) to the appropriate well (except
substrate blank)

6. Conjugate incubation for 30 minutes (+/- 1 min) * at 37°C (+/- 1°C) in moist chamber.

7. After incubation wash all wells with washing solution (see above)

8. Addition of substrate
Add 100 µl substrate solution (ready-to-use) to each well (including well for substrate
blank!)

9. Substrate incubation for 30 minutes (+/- 1 min) * at 37°C (+/- 1°C) in moist chamber.

10. Stopping of the reaction

Add 100 µl stopping solution to each well, shake microtest plate gently to mix.

11. Read optical density

Read OD within 60 minutes at 405 nm against substrate blank, reference wave length
between 620 nm and 690 nm (e.g. 650 nm).

*
Please note, that under special working-conditions internal laboratory adaptations of the incubation
times could be necessary.
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8. TEST EVALUATION
SERION ELISA classic Legionella pneumophila 1-7 IgG/IgM (quantitative)

8.1 Single-point quantification with the 4PL method

Optimized assignment of extinction signals to quantitative values is guaranteed by using

non-linear functions, which adjust a sigmoide curve without any further transformation to
OD-values.

Determination of antibody concentrations with the SERION ELISA classic is carried out by
the logistic-log-model (4 PL; 4 parameter) which is ideal for exact curve-fitting. It is based
on the formula:

D-A
OD = A +
1 + e B(C - In conc.)

The parameters A, B, C, and D are representative for the exact shape of the curve:

1. lower asymptote Ö parameter A

2. slope of the curve Ö parameter B
3. turning point Ö parameter C
4. upper asymptote Ö parameter D
For each lot the standard curve is evaluated by Institut Virion\Serion GmbH (Würzburg,
Germany) in several repeated test runs under optimal conditions. Time consuming and cost
intensive construction of the standard curve by the user is not necessary.

For evaluation of antibody concentrations a lot specific standard curve as well as a lot
specific evaluation table is included with each test kit. Appropriate evaluation software is
available on request.

To compensate for normal test variations and also for test run control a standard serum is
used in each individual test run. For this control serum a ‘’reference value’’ with a validity
range is determined by the quality control of the producer. Within this range a correct
quantification of antibody concentration is ensured. Since the standard serum is not
necessarily a positive control, the value of the standard serum may be borderline or
negative in some ELISA tests.

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8.2 Criteria of validity

- the substrate blank must be OD < 0.25

- the negative control must be negative
- quantitative ELISA: the mean OD-value of the standard serum must be within the
validity range, which is given on the lot specific quality control certificate of the kit
(after subtraction of the substrate blank!)
- qualitative ELISA: the mean OD-value of the positive control must be within the
validity range, which is given on the lot specific quality control certificate of the kit
(after subtraction of the substrate blank!)
- the variation of OD-values may not be higher than 20%.

If these criteria are not met, the test is not valid and must be repeated.

8.3 Calculation
SERION ELISA classic Legionella pneumophila 1-7 IgG/IgM (quantitative)

8.3.1 Non-automated evaluation

For the test evaluation a standard curve and an evaluation table are included in the test kit
so that the obtained OD-values may be assigned to the corresponding antibody activity.
The reference value and the validity range of the standard serum are given on the
evaluation table (quality control certificate).

The blank (A1) must be subtracted from all OD-values prior to the evaluation.

Method 1: Qualitative Evaluation

To fix the cut-off ranges please multiply the mean value of the measured standard-OD with
the numerical data of the certificate of quality control (see special case formulas), e.g.:
OD = 0.502 x MW (STD) with upper cut-off
OD = 0.352 x MW (STD) with lower cut-off
If the measured mean absorbance value of the standard serum is 0.64, the range of the cut-
off is in between 0.225-0.321.

Method 2: Continuous determination of antibody activities using the standard curve.

So called interassay variations (day to day deviations and laboratory to laboratory

deviations) are compensated by multiplication of the current measured value obtained with
a sample with the correction factor F. This factor is calculated as follows:

OD-reference value (of standard serum)

F =
OD-current value (of standard serums)

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The procedure is necessary to adjust the current level of the test of the user with the lot-
specific standard curve.

First, daily deviations have to be corrected by calculating a factor (correction factor F):

1. The mean of the two OD-values of the standard serum has to be calculated and checked
that it is within the given validity range.
2. Calculation of the factor "F": the given reference value is divided by the mean of the
extinction of the standard serum:
F = reference value extinction standard serum / mean value extinction standard serum.
3. All measured values of samples are multiplied by "F".
4. Antibody activities in IU/ml or U/ml can be determined from the standard curve with
the corrected values.

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8.3.2 Automatic test evaluation with
SERION easy base 4PL-Software/SERION evaluate-Software

After input of the 4 parameters and the reference value of the standard serum, antibody
activities are calculated online. If the optical density of the standard is out of the valid
range, the following message will appear:

SERION easy base 4PL-Software:

”Standards are not in tolerance range” and/or “Distance between standards is greater than
20 %.”

SERION evaluate-Software:

“Standard values out of ranges in following groups: Group 1-24. Standard value differ
more than 20 % in following groups: Group 1-24.”

In these cases the test run is invalid and should be repeated.

Parameters and reference value need to be changed only if there is a change of lot
(evaluation table shows parameters and reference values). Correct input of the lot specific
data can be checked on the basis of the IU/ml or U/ml assigned to the standard serum. The
calculated mean value of the units has to correspond to the unit value indicated on the lot
specific certificate. There is an automatic correction of the measured values. In the standard
version the printout displays the following:

sample code
OD-value
IU/ml or U/ml
evaluation

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9. STATEMENTS OF WARNING

9.1 Statements of warning

The SERION ELISA classic is only designed for qualified personnel who are familiar with
good laboratory practice.

All kit reagents and human specimen should be handled carefully, using established good
laboratory practice.

- This kit contains human blood components. Although all control- and cut-off-sera
have been tested and found negative for HBs-Ag-, HCV- and HIV-antibodies, they
should be considered potentially infectious.
- Do not pipette by mouth.
- Do not smoke, eat or drink in areas in which specimen or kit reagents are handled.
- Wear disposable gloves, laboratory coat and safety glasses while handling kit reagents
or specimen. Wash hands thoroughly afterwards.
- Samples and other potentially infectious material should be decontaminated after the
test run.
- Reagents should be stored safely and be unaccessible to unauthorized access e.g.
children.
- Stopping solution: corrosive (C); cause acid burn (R34)
use safety glasses, gloves and laboratory coat while handling!
9.2 Disposal

Please observe the relevant statutory requirements!

10. BIBLIOGRAPHY
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1. Wienieck-Krusnell, J., Linder, E., Free-living amoebae protecting Legionella in water:
the tip of an iceberg? Scand. J. Infect. Dis. 1999, 31(4), 383-385.

2. Helbig, J. H., Lück, P. Ch., Röske, K., und Witzleb, W., Nachweis der intrazellulären
Vermehrung von Legionella pneumophila in Protozoen durch Antigenquantifizierung
mittels ELISA. Zbl. Hyg. 1993, 194, 392-397.

3. Blatt, S. P., Parkinson, M. D., Pace, E., Hoffman, P., Dolan, D., Lauderdale, P., Zajac, R.
A., Melcher, G. P., Nosocomial legionnaires´disease: aspiration as a primary mode of
disease acquisition. Am. J. Med. 1993, 95, 16-22.

4. Neumeister, B., Legionelleninfektionen- Epidemiologie, Diagnostik, Klinik und

Pathogenese. Clin. Lab. 1996, 42, 715-729.

5. Lück, P. Ch., Legionella - eine Herausforderung für das mikrobiologische Labor.

Immun. Infekt. 1993, 21, 3-4.

6. Quigley, C., Legionaires´disease. Microbiol. Europe 1996, 4(3), 10-14.

7. Putzker, M., und Sobe, D., Diagnostik von Infektionen mit Legionella-Spezies. Dtsch.
med. Wschr. 1996, 121, 1608-1615.

8. Cheesbrough, J. S., Makin, T., Taxman, B. C., Beeching, N. J., Mutton, K. J., False-
positive legionella serology in campylobacter infection. The Lancet 1992, 339, 429.

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