The document discusses the extensive process of developing and validating diagnostic ELISA tests. It involves selecting appropriate antigens, determining optimal sample and antigen coating concentrations, establishing calibration curves and cutoff values, and compensating for variability between test lots. Proper development ensures ELISA tests reliably and reproducibly detect antibodies with clinical relevance.

1. Development, Validation and Care
Diagnostics of Infectious diseases with
SERION ELISA classic

2. SERION ELISA classic are CE-certified test systems. Therefore, during
assay development, several harmonized standards must be applied.
This includes documentation and risk assessment. Additionally,
performance evaluation and product stability studies have to be
performed.
SERION ELISA classic

3. The process of assay development and product care is extensive and time
consuming. It comprises several steps of development and verification
studies.
Each immunoassay passes identical development and care steps.
SERION ELISA classic

4. The selection of a distinct antigen is dependent on the clinical demands of
an immunoassay.
For a sensitive detection of acute infections, a maximum number of
epitopes is favorable. This can be achieved by the used of
pathogen preparations.
If there is a risk of detecting cross reactive antibodies, specific antigens
must be selected. The antigens of choice often are preparations of distinct
proteins are recombinant antigens.
Selection of appropriate Antigens

5. For determination of the immune status, only proteins used for vaccination
are suited. Additionally, for several pathogens epitopes are described to
induce protective antibodies.
The various groups of antigens differ among efforts and cost intensities
of their production process.
The cultivation in cell cultures is more expensive than producing
recombinant proteins in bacteria.
Selection of appropriate Antigens

6. For identifications of appropriate antigens,
several antigens are selected and analyzed
concerning their properties.
These analyses include:
• Detection of positive sera
• Exclusion of negative sera
• Exclusion of cross-reactive sera
• P/N ratio
• Homogeneous coating
• Economical characteristics
Selection of appropriate Antigens
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OD
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OD
Patient

7. Selection of appropriate microtiter
Plates
Different antigens exhibit varying properties influencing their binding
capacities to microtiter plates.
Several microtiter plates with different binding capacities are commercially
available.
This plate differ among their specificity of binding
negative, positive or uncharged antigens.

8. Coating of an optimal amount of antigens to the microtiter plates ensures
the reproducible and reliable results.
Microtiter plates have a maximum binding capacity. The antigen
concentration should not fall below or exceed the binding capacity as it
strongly influenced the assay‘s results.
Coating concentrations
saturated saturation exceeded
unsaturated

9. Coating concentrations
With increasing binding concentrations the achieved signal will be
enhanced. Around the concentration of antigen saturation, a plateau will be
reached.
With further increasing concentrations, the signal will drop (Hook effect).

10. Coating Conditions
Next to the antigen concentrations, coating conditions such as temperature
and duration influence the immunoassay‘s performance.
Blocking solutions bind to uncoated areas of the
plate and stabilize bound antigens.
With blocking
Without blocking

11. Determination of Sample Dilution
In samples with high antibody concentrations low serum dilutions can lead
to unspecific analyte binding resulting in false-positive results.
Additionally, interfering substances can influence
antibody-antigen interactions.
These influences are known as matrix effects and are mainly caused by
divalent ions (Mg2+ or Ca2+).
The higher the chosen serum dilution,
the lower the influence of the serum matrix.

12. Determination of Sample Dilution
Selection of a very high serum dilution impairs the sensitive detection of
acute infections.
Additionally, high sample dilutions can reduce the P/N ratio and therefore
reduce precision of sample evaluation.
As calibration curves will be
flattened the range antibody
quantification is reduced and
U/ml values are less reliable.

13. Determination of Sample Dilution
The majority of ELISA manufacturers set serum dilutions to 1+100. This
dilution usually ensures an optimal balance between reducing interfering
substances and signal intensities.
For detection of antibodies specific for parameters of high seroprevalence,
the selection of higher dilutions is suited for the
compensation of seroprevalence.
Increased dilutions go along with higher detection limits.

14. Antibody Quantification
With antibody quantification further information about a disease can be
received.
Determination of antibody activities allow for disease staging,
monitoring of titer changes and therapy control.
Additionally, determination of immune status, vaccination control or
drawing of vaccination recommendation is possible.
For CSF diagnostics, a reliable antibody quantification is a basic demand.

15. Antibody Quantification
For quantification of results obtained with SERION ELISA classic a
„Stored Master Curve“ is prepared during the quality control process of
each lot. This quantification curve is valid over the product‘s life-cycle.
For generating a quantification curve serum samples with
high antibody activity are needed.
For some pathogens international standard preparations with defined
antibody activities are available.
If there is no standardized calibration material available, a company-own
standard serum will be used. This samples we be assigned to a defined
antibody activity (= „arbitrary Units“).

16. Antibody Quantification
A calibration serum is diluted linearly in 10 - 15 steps. Blotting the OD in
correlation to the dilution results in a sigmoidal curve. Each step can be
assigned to the predefined U/ml values.
This calibration curve is determined in several runs under optimal
conditions.
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U/ml
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OD
Dilution steps

17. Antibody Quantification
In order to determine a mathematical function for activity calculation, a
curve fit based on the 4PL method is performed. A program varies the
parameters A, B, C and D in order to find on optimal fit to the
experimentally determined data.
Activity (U/ml) = 𝑒
𝐶−
1
𝐵
ln(
𝐷−𝐴
𝑂𝐷 𝑃𝑎𝑡𝑖𝑒𝑛𝑡 ∗𝐹 −𝐴
−1)
A = lower asymptote
B = slope
C = infliction point
D = upper asymptote
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U/ml
D
A
C
B

18. Calibration
In order to compensate for run-to-run deviations, a correction has to be
performed. By this correction, the achieved measurement values are
referred to the quantification curve.
The standard serum serves as a
calibrator.
The target value is determined
during quality control under
optimal conditions.
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OD
U/ml

19. Calibration
The standard serum should be assigned to a target value of
OD = 0.7 – 1.0. This OD-region is within the pseudolinear range of the
standard curve.
In order to identify a serum with the desired criteria,
a high number of sera is analyzed.
Standard sera are composed of one single serum sample.
Pooled or spiked serum samples do often not yield reproducible results.
The standard serum must be available in sufficient amounts.

20. Negative control
Similar to the standard serum, also negative controls are composed of only
one single serum sample.
The sample must should be evaluated as negative homogeneously
In order to identify a serum with the desired criteria, a high number of sera is
analyzed.

21. The borderline ranges serves for serum evaluation in „positive“, „negative“
or „borderline“.
The more sensitive a test system is set, the more false-positive results
will occur (reduced specificity).
Increasing the borderline range in order
to enhance specificity will
reduce sensitivity.
Determination of borderline ranges
Number
of
spamples
U/ml
specific
sensitive
negative panel positive panel

22. Determination of borderline ranges
The most simple way to determine borderline ranges is to use
only negative serum samples.
The borderline is determined by calculating a mean value and adding
the 2- or 3-fold standard deviation.
This results in an
analytical cut off.
It is the most sensitive cut-off
that can be achieved.
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OD
Serum Nr.
MW
3δ

23. Determination of borderline ranges
Sample evaluation with SERION ELISA classic is most often based on a
„clinical cut-off“.
This borderline range is determined by a panel of healthy blood donors
and patients with acute infections.
This kind of cut off allows for the
detection of antibodies with
clinical relevance only.
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Serum Nr.

24. Determination of borderline ranges
For some detection systems for antibodies against pathogens with
high seroprevalences, the results can be evaluated with different cut offs.
The lower borderline range serves
for the sensitive detection of acute
primary infections in children.
The higher cut off allows for
compensation of seroprevalence.
It can be used for analysis of
samples derived from patients older
than 4 years of age.

25. Conjugate Solutions
Minor modifications of single assay components can influence the test
level.
Components of biological origin such as antigens can vary among their
composition between different lots.
As a consequence, different epitopes can be expressed in varying
extends.
This can result in higher or lower OD-values when comparing assays
based on different antigen lots.

26. Conjugate Solutions
Antigens with high activity can induce limitations close to the
upper measurement range (see a).
Lower activities result in a calibration curve with low amplitude. The
quantification is less reliable and limited to a smaller measurement range
(see b). The discrimination between positive and negative results is
impaired.
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OD
U/ml
a)
b)
U/ml

27. Conjugate Solutions
In order to compensate for these different test levels and to keep similar
measurement results over several kit lots, signal intensities can be
amplified or reduced.
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OD
U/ml U/ml

28. Conjugate Solutions
For compensation of these deviations, each lot of SERION ELISA classic is
assigned to conjugate solutions of different strength.
These conjugates differ among their concentrations of the detection
antibody.
Lots with lower activity are used with higher concentrated conjugates
solutions and the other way around.
+ ++ +++

29. Conjugate Solutions
A change of conjugate solution strength only influences the level of
measurement values.
Antibody activities (U/ml) or serum sample evaluations are not affected.
SERION ELISA classic
Diphtheria IgG
Lot SEE.BD
(++) Conjugate Solution
SERION ELISA classic
Diphtheria IgG
Lot SLE.BN
(+++) Conjugate Solution
Serum sample OD IU/ml OD IU/ml
SIP.65 1.322 > max 1.723 > max
K18-L 0.814 0.73 1.093 0.70
SIP.68 0.197 0.10 0.266 0.10
K149-L 0.134 0.07 0.186 0.06
SHM.14 0.520 0.36 0.784 0.39
SHM.15 0.066 < min 0.097 < min
K17-L 0.296 0.17 0.42 0.16
SKQ.17 1.463 > max 1.907 > max

30. Dilution Buffer
For the majority of SERION ELISA classic test systems an identical dilution
buffer is used. This enables easy handling and automated processing.
The buffer is based on a buffered saline solution with a defined pH-value
and contains a protein stabilizer.
In order to achieve increased specificities, for some SERION ELISA classic
special dilution buffers will be used.
These buffers serve for absorption of
cross-reactive antibodies.

31. Performance Evaluation Studies
SERION ELISA classic

32. Performance Evaluation
The performance evaluation is done for every SERION ELISA classic in
accordance to DIN ISO 13612.
During these studies the following analyses are made:
Sensitivity and specificity
Measurement range
Precision
Linearity
Cross-reactions
Interfering substances

33. Determination of
Sensitivity and Specificity
For determination of sensitivity and specificity a panel of predefined serum
samples from patients and healthy blood donors is analyzed.
Performance parameters are calculated in comparison to other
immunoassays. For a reliable determination, several comparative assays
should be used.

34. Determination of
Sensitivity and Specificity
Sensitivity is determined with serum samples that are evaluated as positive
with the comparative assay.
Specificity is determined by negative serum samples.
Sensitivity =
Specificity =
true positive
true positive + false negative
true negative
true negative + false positive
Comparative assay

35. Determination of
Measurement Ranges
Close to the upper or lower asymptotes of the quantification curve, slight
deviations of measurement values will lead to extensive deviations in
the determined antibody activity.
The obtained U/ml values are not reproducible.
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OD
U/ml
Δ
Δ
Δ
Δ

36. Determination of
Measurement Ranges
Due to insufficient precision the measurement ranges are limited.
The coefficient of variation (CV) is used to determine the precision of
antibody quantification. The CV is calculated by the quotient of standard
deviation and the mean value of repeated analyses.
The measurement range
is limited to regions with
a CV < 20 %.
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U/ml
CV
[%]

37. Intraassay precision
The precision of SERION ELISA classic is determined within one
microtiter plate.
For this purpose sera of different antibody activities are analyzed at
different positions of a microtiter plate.
The precision is determined by
the CV among each sample.
It is an indicator of the
homogeneity of antigen coating.
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1 1
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1
1

38. Interassay precision
In order to determine the precision among several microtiter plates sera
of different antibody activities are analyzed on 10 plates.
The precision is determined by
the CV among each sample.
It is an indicator of the
homogeneity of antigen coating.

39. Thank you!