Dorobantu_A_Luminex-bead-based assay - Feedback

1. Coursework Template for E-submission
BMS
Student name Adina Georgiana Dorobantu
Student number 30019475
Module name Biomedical Investigations
Title of coursework Luminex bead-based essay
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2. Technique based essay:
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Luminex bead-based assay
Scientific Theory
Since it was first discovered and introduced in 1995, the Luminex bead-based assay has revolutionized
the approach of scientists in both research and clinical settings, due to the great advantages it brought to
already existing techniques, the most common of which being the ELISA (Bourdon, 2016). Therefore, the
objective of this technique guide is to create a comparative analysis between these two similar assays, while
emphasizing the improvements brought on by Luminex through xMAP Luminex technology (see Table 1).
The multiplex Luminex technology has been developed starting from and improving the concept of
single-plex `sandwich` ELISA (can only detect one analyte at a time), where the antigen is caught between two
different antibodies, one recognizing the antigen and one used for its detection of binding and therefore, its
specific and sensitive quantification (“What is a Luminex Assay?,” 2017). Following the same concept, the
principle behind xMAP Luminex technology is based on the use of color-coded 5.6 µm beads, which have
been impregnated with different ratios of infrared and red dyes (fluorophores), allowing subsequent detection
of unique bead regions specific for certain analytes, that can be identified on the resulting spectra (Principle of
the Luminex Multiplex Bead Immunoassay Method, n.d.).
Additionally, each bead is further coated with specific capture antibodies (Figure 1) (“What is a
Luminex Assay?,” 2017). The multiplex feature of the Luminex assay comes with the advantage of being able
to mix multiple specific beads depending on the required sample for analysis, in order to test for multiple
analytes simultaneously, in the same amount of resources, such as sample volume, incubation time, reagent
amounts, cost (“What is a Luminex Assay?,” 2017).
The sample is added and left to incubate with the bead mixture, ensuring maximal binding and
antibody saturation with analytes of interest, followed by a series of washing steps that ensure elimination of
any unbound materials (Principle of the Luminex Multiplex Bead Immunoassay Method, n.d.). Then, a second
biotinylated antibody linked to a streptavidin-phycoerythrin (PE) fluorescent reporter, also known as the
detection antibody, is added to the mixture and incubated (Figure 1) (Principle of the Luminex Multiplex Bead
Immunoassay Method, n.d.).
The next step of the Luminex assay is the two-way flow cytometry detection system: firstly, a red laser
is used for the excitation of the bead region, together with the bound analyte, respectively; secondly, a second
green laser is used to determine the PE fluorescent reporter signal, which can also be correlated as having a
directly proportional relationship to the amount of bound analyte (Figure 1) (“What is a Luminex Assay?,”
2017). In other words, the more analyte detected by the capture antibodies on certain bead-regions, the
stronger the fluorescence signal, and therefore, the higher the value read by the xMAP Luminex analyser
(“What is a Luminex Assay?,” 2017). Alternatively, measurement of this assay can also be done using the
LED system (“What is a Luminex Assay?,” 2017).
As an immunoassay, xMAP Luminex technology is currently being used in order to measure the
`amount` of various analytes, such as cytokines, chemokines and other biomarkers, mostly present during an
immunological response, which are essential in the better understanding of diseases and their manifestations
(Günther, Becker, Göpfert, Joos, & Schneiderhan-Marra, 2020).

3. For the purpose of the following and future analysis, it should be mentioned that variability is to be
expected between different Luminex assay and ELISA kit producers.
Figure 1. The mechanism behind the Luminex bead-based assay. This diagram outlines the successive steps
of the Luminex assay. It depicts the Luminex bead (shown in red as a suggestion to the dyes used) with its
capture antibody linked to it, which recognized the analyte of interest. Furthermore, the detection antibody
with the linked PE reporter is also observed, as well as the overall bound complex, which is then measured by
the use of red and green lasers (as seen on the right side of the diagram) Diagram retrieved from (Bourdon,
2016).
Luminex bead-based assay ELISA
Analyte capacity Multiplex Single-plex
Ease of use  More accessible and easy
to use
 Easily adjustable
(duPont, Wang, Wadhwa,
Culhane, & Nelson, 2005)
 Labour intenstive
(duPont, Wang, Wadhwa, Culhane,
& Nelson, 2005)
Sample size  25 - 50 µL/ multiple
samples
 This directly influences
and significantly lowers
the cost of the assay
(Mountjoy, 2020)
 50 - 100 µL/ sample
(recommended)
(Mountjoy, 2020)
Coating antibody
(capture antibody)
amount needed
(/well)
 7.5 ng (over 50x the
amount needed for
ELISA/well)
 This is also reflected in a
lower overall assay cost
(Baker, Murphy, Lopez, &
Garcia, 2012)
 400 ng
(Baker, Murphy, Lopez, & Garcia,
2012)

4. Accuracy  Similar or equivalent for
most analytes
 However, this can be
overcome by higher
numbers of measured
analytes in a shorter
period of time
(Shapiro, Wang, Mendu & Firpo,
2020)
 Similar or equivalent
 Slightly more accurate for a
small number of analytes
(Shapiro, Wang, Mendu & Firpo,
2020)
Sensitivity  <4 pg/mL
(Baker, Murphy, Lopez, &
Garcia, 2012)
 >31 pg/mL
(Baker, Murphy, Lopez, & Garcia,
2012)
Cross reactivity Low chance High chance
Specificity and
affinity
 Optimal at the
appropriate parameters
for the analyzed sample
(duPont, Wang, Wadhwa,
Culhane, & Nelson, 2005)
 Cross-reactivity can
interfere
(“Overview of ELISA | Thermo
Fisher Scientific - UK,” 2022)
Detection range  <4 pg/mL to >8000
pg/mL
 Allows a broader range to
be detected by the plate
reader, between OD 3 or
4
(Baker, Murphy, Lopez, &
Garcia, 2012)
 16 pg/mL to 1000 pg/mL
 Could restrict the plate
reading ability due to the
low upper limit
(Baker, Murphy, Lopez, & Garcia,
2012)
Time  ≤ 3h
 Translates to higher
throughout
(Baker, Murphy, Lopez, &
Garcia, 2012)
 ≥ 24h
 Translates to lower
throughput
(Baker, Murphy, Lopez, & Garcia,
2012)
Cost Around 30% less compared to
ELISA, due to smaller amounts
of material needed for analysis of
multiple analytes (£)
(duPont, Wang, Wadhwa,
Culhane, & Nelson, 2005)
Higher cost, due to higher amount
of materials needed for analysis of
multiple analytes (£££)
(duPont, Wang, Wadhwa, Culhane,
& Nelson, 2005)
Type pf plate and
binding surface area
 Carried out in 96-well or
384-well plates
 Due to the beads being
held in suspension, less
capture antibody is
required, as well as a
lower sample amount
(Baker, Murphy, Lopez, &
Garcia, 2012)
 Usually carried out in a 96-
well or 384-well
polystyrene plates
 The flat-surface used
requires increased capture
antibody for coating the
wells and a corresponding
larger sample amount
(Baker, Murphy, Lopez, & Garcia,
2012)
Table 1. Comparative analysis between the Luminex bead-based assay and ELISA

5. Applications
A recent study “Evaluating SARS-CoV-2 spike and nucleoplasmid proteins as targets for antibody
detection in severe and mild COVID-19 cases using a Luminex bead-based assay (Mariën et al., 2021) can be
considered a very good example that proves the high and continuously developing applicability of this
technique. The paper describes the context of choosing the Luminex bead assay, in the detriment of the already
existing ELISA test that however, due to the high demand, is slowly becoming increasingly harder to sustain,
financially, but also disadvantageous and inaccurate over a long period of time, because of suboptimal
specificity and single-plex mechanism (Mariën et al., 2021).
Moreover, the high accuracy, specificity and sensitivity of multiplex Luminex technology measuring
the spike (S) and nucleoplasmid (NP) analytes was carried out on a diversity of recent and old infection cases,
as well as sever, mild and asymptomatic cases (Mariën et al., 2021).
The combination of two SARS-CoV-2 analytes as part of the multiplex Luminex assay generated
considerably higher specificity, that could be used in future test developments, for example, to be distributed in
underdeveloped countries, such as the sub-Saharan Africa, where occurrence of false-positive ELISA tests is
elevated due to the influence of other infectious diseases (Mariën et al., 2021).
The current relevance of this study demonstrates the reliability of the xMAP Luminex technology in
easily adapting to development of novel diagnostic tests, even for a rapidly spreading infectious disease, which
could only encourage an increasing interest in future diversification of this technique.

6. Reference List
Baker, H. N., Murphy, R., Lopez, E., & Garcia, C. (2012). Conversion of a Capture ELISA to a Luminex xMAP Assay using a
Multiplex Antibody Screening Method. Journal of Visualized Experiments, (65). https://doi.org/10.3791/4084
Bourdon D. (2016, May 19). LuminexTM
bead-based immunoassays drive immunoassays towards higher-content biomarker
discovery. Retrieved February 3, 2022, from Behind the Bench website:
https://www.thermofisher.com/blog/behindthebench/luminex-bead-based-immunoassays-drive-immunoassays-
towards-higher-content-biomarker-discovery/
duPont, N. C., Wang, K., Wadhwa, P. D., Culhane, J. F., & Nelson, E. L. (2005). Validation and comparison of luminex multiplex
cytokine analysis kits with ELISA: Determinations of a panel of nine cytokines in clinical sample culture
supernatants. Journal of Reproductive Immunology, 66(2), 175–191. https://doi.org/10.1016/j.jri.2005.03.005
Günther, A., Becker, M., Göpfert, J., Joos, T., & Schneiderhan-Marra, N. (2020). Comparison of Bead-Based Fluorescence
Versus Planar Electrochemiluminescence Multiplex Immunoassays for Measuring Cytokines in Human
Plasma. Frontiers in Immunology, 11. https://doi.org/10.3389/fimmu.2020.572634
Mariën, J., Ceulemans, A., Michiels, J., Heyndrickx, L., Kerkhof, K., Foque, N., … Ariën, K. K. (2021). Evaluating SARS-CoV-2
spike and nucleocapsid proteins as targets for antibody detection in severe and mild COVID-19 cases using a
Luminex bead-based assay. Journal of Virological Methods, 288, 114025.
https://doi.org/10.1016/j.jviromet.2020.114025
‌
Mountjoy K.G. (2020) ELISA versus LUMINEX assay for measuring mouse metabolic hormones and cytokines: sharing the
lessons I have learned. Retrieved February 3, 2022, from Journal of Immunoassay and Immunochemistry website:
https://www.tandfonline.com/doi/full/10.1080/15321819.2020.1838924
Overview of ELISA | Thermo Fisher Scientific - UK. (2022). Retrieved February 3, 2022, from Thermofisher.com website:
https://www.thermofisher.com/uk/en/home/life-science/protein-biology/protein-biology-learning-center/
protein-biology-resource-library/pierce-protein-methods/overview-elisa.html#:~:text=The%20enzyme%20linked
%20immunosorbent%20assay,microplate%20wells%20using%20specific%20antibodies
PRINCIPLE OF THE LUMINEX MULTIPLEX BEAD IMMUNOASSAY METHOD. (n.d.). Retrieved from https://cdn
links.lww.com/permalink/sla/b/sla_2017_07_03_allen_annsurg-d-17-00426_sdc1.pdf (pp. 3, 7, 12-13)

7. Shapiro, M S; Wang, X; Mendu, D R; Firpo (2020) Multiplex Assays of Luminex for Identification of COVID-19 Antibodies in
Patient Serum: Performance Evaluation and Comparison to ELISA, A.American Journal of Clinical Pathology, suppl.
1; Chicago Vol. 154, (Oct 2020): S92. DOI:10.1093/ajcp/aqaa161.201, Retrieved February 3, 2022, from
Proquest.com website: https://www.proquest.com/openview/d6c8b8d6d42fd46a252b18220b5f4213/1?pq-
origsite=gscholar&cbl=586299
What is a Luminex Assay? (2017). Retrieved February 3, 2022, from www.rndsystems.com website:
https://www.rndsystems.com/what-luminex-assay

8. Faculty of Health and Wellbeing - Department of Biosciences
ASSESSED WORK FEEDBACK FORM
Student Name:
Module Title: Biomedical Investigations
Title of coursework Technique based structured essay
Marker:
Grade
Strengths:
Weaknesses and suggestions for improvement
Student comments for Feed-forward (how will you use this feedback to improve your future work?):
SIGNATURE DATE:
*A provisional mark may be raised or lowered by the External Examiner or the Exam Board

9. Indicator First
(Exceptional)
First (Excellent) Upper second
(Very good)
Lower Second
(Good)
Third
(Sufficient)
Fail
(Insufficient)
Low/Zero Fail
Overall
Knowledge and
Understanding
(weighting
20%)
Shows advanced
understanding of key
subject area. Excellent
level of originality with
a wide range of
relevant literature
accessed.
Excellent grasp of
relevant literature and
sound understanding
of subject area.
Evidence of originality.
Very good
understanding of the
area. Appreciation of
wider implications,
and a serious attempt
to engage with
breadth of relevant
literature
Good understanding
of subject area.
Evidence of a
reasonably sound
engagement with
relevant literature.
May contain minor
errors.
Sufficient or
inconsistent grasp of
material. Evidence
of some
understanding of
subject area.
Limited research
and major errors.
Insufficient. Partial
answer with major
omissions. Poor
understanding.
Scarce amount of
research.
Little or no attempt to
address the question.
Brief or wholly irrelevant
answer.
Scientific
theory with
diagram
(weighting
30%)
Outstanding scientific
theory. Clearly and
concisely written.
Flows well. Excellent
diagram with clear
explanatory legend
Excellent theory, Clear
and coherent.
Excellent diagram with
appropriate legend
Very good theory.
Clear and coherent.
Clear diagram with
appropriate legend
Good explanation of
theory. Diagram
included. Figure
legend included
Sufficient
explanation. Lacks
coherence and
clarity in many
areas. Poor choice
of diagram. Legend
does not explain
figure well.
Insufficient theory.
Explanation lacks
focus. Incoherent,
and lacks clarity
throughout. Poor
diagram, legend
missing or irrelevant.
Very poor with no
structure or focus.
No attempt at figure or
figure legend
Table of
comparison to
another
technique
(weighting
20%)
Outstanding critical
analysis.
Excellent use of
literature to illustrate
comparisons
Excellent standard of
intelligent, critical
thought and argument.
Good use of literature
to illustrate
comparisons
Very good evidence
of independent
thinking and critical
analysis. Relevant
material.
Good evidence of
critical analysis
Limited use of
literature to illustrate
comparisons
Sufficient/ weak
critical analysis. An
attempt to answer
the question but
may lack relevance
and very little use of
literature
Unfocused.
Insufficient
engagement with
literature
No understanding of the
issues and little attempt to
address them
Application of
use
(weighting
20%)
Outstanding account of
application discussed.
Accurately
communicated. Benefits
fully explained
Excellent
understanding of the
applications. Highly
relevant applications
incl. Clearly and
effectively written.
Benefits well explained
Very good
understanding of the
applications. Relevant
choices with some
appropriate critique
Benefits explained
Good understanding
of the applications
discussed. Relevant
choices made,
benefits included
Sufficient/ limited
explanation of
applications of use,
unfocused and
lacking depth.
Insufficient attempt
to explain
applications,
sources of
information poor or
inappropriate
Little or no attempt to
discuss applications of
use
Referencing
(weighting
10%)
Excellent and
appropriate use of
referencing
throughout. No errors.
Outstanding reference
choice throughout
Refs correct and
thorough. Bibliography
complete- properly laid
out. Very minor errors.
Excellent ref choice
throughout
References accurate.
Bibliography
complete and
properly laid out.
Minor errors. Very
good ref. choice
Generally correct
but needs some
attention.
Some good
reference choices
Some incorrect
referencing and
incomplete or not
properly laid out
bibliography.
Insufficient or no
proper referencing.
Bibliography
inadequate. Poor
choice of references
No referencing
Class CG% General Characteristics Level 5
9

10. FIRST
(Excellent}
96 Exceptional breadth and depth of knowledge and understanding of the area of study; evidence of extensive and appropriate selection and critical
evaluation/synthesis/analysis and of reading/research beyond the prescribed range, in both breadth and depth, to advance work/direct
arguments; exceptional demonstration of relevant skills; excellent communication; performance deemed to be beyond expectation.
89
81 Outstanding/excellent knowledge and understanding of the area of study as the student is typically able to go beyond what has been taught
(particularly for a mid/high 1st
); evidence of extensive and appropriate selection and critical evaluation/synthesis/ analysis of reading/research
beyond the prescribed range, to advance work/direct arguments; excellent demonstration of relevant skills; excellent communication;
performance deemed beyond expectation of the level.
74
UPPER SECOND
(Very good)
68 Very good knowledge and understanding of the area of study as the student is typically able to relate facts/concepts together with some
ability to apply to known/taught contexts; evidence of appropriate selection and evaluation of reading/research, some beyond the prescribed
range, may rely on set sources to advance work/direct arguments; demonstrates autonomy in approach to learning; very good
demonstration of relevant skills; strong communication skills.
65
62
LOWER SECOND
(Good)
58 Good knowledge and understanding of the area of study balanced towards the descriptive rather than analytical; evidence of appropriate selection
and evaluation of reading/research but generally reliant on set sources to advance work/direct arguments; good demonstration of relevant skills,
though may be limited in range; communication shows clarity but structure may not always be coherent.
55
52
THIRD
(Sufficient)
48 Knowledge and understanding is sufficient to deal with terminology, basic facts and concepts but fails to make meaningful synthesis; some
ability to select and evaluate reading/research however work may be more generally descriptive; strong reliance on available support set sources
to advance work; arguments may be weak or poorly constructed; adequate demonstration of relevant skills over a limited range;
communication/presentation is generally competent but with some weaknesses.
45
42
FAIL
(Insufficient)
35
Insufficient knowledge and understanding of the area of study; some ability to select and evaluate reading/research however work is more
generally descriptive; fails to address some aspects of the brief; a limited use of sources to advance work; arguments may be weak/poor or
weakly/poorly constructed; demonstration of relevant skills over a reduced range; communication shows limited clarity, poor presentation,
structure may not be coherent.
25
15
Highly insufficient knowledge or understanding of the area of study; understanding is typically at the word level with facts being reproduced in a
disjointed or decontextualised manner; fails to address the outcomes addressed by the brief; typically ignores important sources in development of
work and data/evidence inappropriately used; weak technical and practical competence hampers ability to demonstrate/communicate achievement of
outcomes.
5
Zero 0 Work of no merit OR absent, work not submitted, penalty in some misconduct cases.
10