Indirect ELISA Test: Procedure, Results, and Discussion

The report discusses the procedure, results, and discussion of an indirect ELISA experiment performed to detect human chorionic gonadotropin (hCG) in serum or urine. It includes calculations of mean, standard deviation, and standard error of mean. The report also highlights the importance of quality controls and pre-analytical precautions in ELISA testing.

Indirect ELISA test1
INDIRECT ELISA TEST
Name:
Department:
School:
Date:

Indirect ELISA test 2
Abstract
The report aims to perform an indirect ELISA experiment. The basic approaches
comprise immobilising an antigen onto a compact surface and then compiling it with antibodies.
Binary antibodies are utilised in this experiment. First is the primary antibody that fixes to the
antigen and second is a secondary antibody that joins to the primary antibody. The secondary
antibody is conjugated to an enzyme. Finding was done by growth with an enzyme substrate that
produced a specific colour. In the material and methods section, serial dilutions were done from
the stock solution. After the experiment, it was expected to comprehend the concepts and
procedure of an indirect ELISA, how to develop a standard curve using Microsoft Excel, and
how to determine unknown’s samples from the graph.

Indirect ELISA test 3
Introduction
Human chorionic gonadotropin (hCG) is usually a glycoprotein hormone made during
pregnancy in uterus. The hCG fragments comprise of twofold joined and having different
subdivision labelled as alpha and beta. The alpha subunit, with a molecular weight of about
18000 Daltons is same to alpha subunits of the pituitary glycoprotein hormones such as thyroid-
stimulating, luteinizing and follicle stimulating hormone (Salvati et al. 2013). On the other side,
beta subunits deliberate on genetic and immunological specificity to the complete HCG particle
by virtue of its distinctive amino acid series and constituents (Okda et al. 2015).
The outlook of hCG in serum or urine soon after conceiving and its quick increase in
strength makes it a perfect sign for the finding and validation of pregnancy (Choi and Smitz
2014). But, raised hCG intensities are also often linked with non-trophoblastic and trophoblastic
neoplasm. Other case is where women have multiple pregnancies, and thus, intensities of hCG
are recounted being higher than those anticipated during a usual single pregnancy. Additionally,
lower hCG has been linked with the threatened abortion and ectopic pregnancy (Ezcurra and
Humaidan 2014).
ELISAs are important in detecting peptide hormones such as hCG as it is formed in large
sums by a developing placenta during pregnancy which forms the foundation of numerous
pregnancy tests (Youssef et al. 2014). HCG may also be made unusually by particular tumours
precisely from the egg or sperm. HCG levels are frequently used in female to test the atypical
tissue in their uterus, molar pregnancy or tumor in the uterus (choriocarcinoma) rather than usual
pregnancy (Gan and Patel 2013).

Indirect ELISA test 4
Materials and methods
100 ul of hCG antigen (50mg/ml) was put to well 1 and 50ul of PBS was added to wells 2
to 9 in rackets. 50ul of hCG was taken from well 1 and added and mixed appropriately in well 2.
After blending, 50ul from well 2 was put to well 3 and mixed thoroughly. Serial dilution was
continued up to well 8. 50ul from well 8 was taken and discarded into the water glass and it was
not added to well 9. After serial dilutions, 50ul of patient serum sample A was put into well 10,
50ul of patients urine sample B was put into well 11, and finally, 50ul of patient urine sample C
was added into well 12. Also, repeated stages from 1 to 8 in rows of sample B and C, so that
there could be 3 replications (Kuang et al. 2013). 96 well plates were developed at room
temperature for 15 minutes. The wells were washed by adding 100ul PBS/Tween to each well,
mixing and the liquid removed using a pipette set at 150ul. The wells were washed threefold and
50ul of primary antibody was put to every well and incubated. 50ul of secondary antibody was
put to all well and gestated for each well and incubated for 5-10mintes till the colour was
established. The reaction was clogged by addition of 50ul of 2M sulphuric acid. The reading was
done by a plate reader at an absorbance of 450nm (Song, Sun, Wang, Nai and Liu 2014).
Results
Calculation
Well 1= antigen concentration = (50mg/ml)
Well 2 Initial Volume 50ul
Final volume 100ul
Therefore, serial dilution factor=50ul/100ul=1/2 dilution factor

Indirect ELISA test 5
Thus, the concentration in well 2=50mg/ml/2=25mg/ml
Well 3=25mg/ml*dilution factor=12.5mg/ml
Well 4=12.5mg/ml*dilution factor=6.25mg/ml
Well 5=6.25mg/ml*dilution factor=3.125mg/ml
Well 6=3.125mg/ml*dilution factor=1.5625mg/ml
Well 7=1.5625mg/ml*dilution factor=0.781mg/ml
Well 8=0.781mg/ml*dilution factor=0.391mg/ml

Indirect ELISA test 6
Absorbance at 450nm
concentration hCG (mg/ml) A B C
50 0.219 0.215 0.186
25 0.211 0.189 0.189
12.5 0.214 0.182 0.177
6.25 0.192 0.188 0.205
3.125 0.188 0.168 0.22
1.5625 0.185 0.163 0.226
0.781 0.181 0.174 0.233
0.391 0.179 0.162 0.209
0.391 0.181 0.162 0.259
0 2 4 6 8 10 12
0
2
4
6
8
10
12
f(x) = NaN x + NaNf(x) = NaN x + NaNf(x) = NaN x + NaN Graph of absorbance Vs concentration
Concetration (mg/ml)
Absorbance
third set
second set
first set
Figure 1: graph showing all raw data of absorbance and concentration in three data sets

Indirect ELISA test 7
Calculations of mean (average), standard deviation (SD) and standard error of mean
(SEM)
1 2 3 4 5 6 7 8 9 10 11 12
A 0.219 0.211 0.214 0.192 0.188 0.185 0.181 0.179 0 0.175 0.181 0.182
B 0.215 0.189 0.182 0.188 0.168 0.163 0.174 0.162 0 0.16 0.193 0.165
C 0.186 0.189 0.177 0.205 0.22 0.226 0.233 0.209 0 0.224 0.28 0.223
average 0.206667 0.196333 0.191 0.195 0.192 0.191333 0.196 0.183333 0 0.186333 0.218 0.19
st.dev 0.018009 0.012702 0.020075 0.008888 0.02623 0.031974 0.032234 0.023798 0 0.033471 0.054028 0.029816
SEM 0.010398 0.007333 0.01159 0.005132 0.015144 0.01846 0.01861 0.01374 0 0.019325 0.031193 0.017214
Standard deviation calculates the dispersion of the data around the mean (Wan, Wang,
Liu and Tong 2014). Low standard deviation means the data are close to the mean and therefore
lower SD means that data has a lower degree of variability. Higher SD means that the data as
high degree of variability. Standard error mean (SEM) quantify the precision of the mean and
measures how far the sample mean is to the true mean of calculation. Low SEM denotes that
calculated mean is close to the actual mean. Therefore, the SEM measures the precision of data
(Wan, Wang, Liu and Tong 2014). Basing above argument on the data presented, it is clear that
there is lower standard deviation. This translates that there is a lower degree of viability and
hence high precision.

Indirect ELISA test 8
absorbance (450nm)
concentration (mg/ml) 50 0.207
25 0.196
12.5 0.191
6.25 0.195
3.125 0.192
1.5625 0.191
0.781 0.196
0.391 0.183
0 2 4 6 8 10 12
0
2
4
6
8
10
12
f(x) = NaN x + NaN
Graph of absorbance (nm) vs concentration
(mg/ml)
concentration (mg/ml)
absorbance (nm)
mean absorbance
Calculation of the sample A, B and C
The slope of the graph is y=mx+c
Where y=absorbance (with no units)
M=slope of the graph (molar extinction coefficient)
C= y intercept
X=concentration of the sample

Indirect ELISA test 9
Therefore, sample A with absorbance of 0.186
0.186=-0.02x+0.203
X=0.85mg/ml
Sample B with absorbance of 0.2181
0.2181=-0.02x+0.203
X=-0.755mg/ml
Sample C with absorbance of 0.190
0.190=-0.02x+0.203
X=0.65mg/ml
Discussion
Good laboratory routine necessitates that quality controls be done with all calibration
curve to confirm assay routine. To ensure suitable procedure, a statistically considerable amount
of controls ought to be assayed to determine mean values and satisfactory series (Santibañez et
al. 2014). The dependable and reproducible result will be got when the assay process is done
with a comprehensive thoughtful of the package guidelines and the compliance to apt laboratory
routine. The data found from the usage of this set ought to be used merely as an aide to other
investigative trials and the info accessible to the doctor (Zarei et al. 2014). The wash process is
dire; therefore, inadequate washing will result to poor precision and incorrectly higher
absorbance interpretations. It worth noting, a normal pregnancy cannot be differentiated from an

Indirect ELISA test 10
ectopic pregnancy grounded on the HCG level only (Křišt’an, Alavi, Stejskal and Policar 2013).
Also, impulsive miscarriage may result in mix-up in understanding test results.
In pre-analytical precautions, every laboratory must institute its own regular series reliant
on the patient populace. HCG is not usually discovered in the serum of well non-pregnant
females and healthy men. The strength of hCG in the serum of expectant women upsurges to 5-
50mlu/ml one week after implantation and remains to increase steadily during the ten weeks,
getting to a maximum of 100-00,00mlU/ml at the finale of the first trimester (Bourdiec, Calvo,
Rao and Akoum 2013). Though regular pregnancy is commonly the cause of augmented hCG
intensities in serum and urine, raised hCG strength has also been testified in patients detected
with non-trophoblastic neoplasms, choriocarcinoma and molar pregnancy. Therefore, further
testing is recommended to ascertain the proper diagnosis (Kitahara, Nakamura, Kogure and
Minegishi 2013).
In manual pipetting, it is suggested that not more than 32 wells be applied for every
single assay run. Pipetting of entirely standard samples and control should be done within three
minutes. In the automatic pipetting, a complete plate of 96 well may be applied in an easy assay
run (Huang et al. 2016). But, it is commended that pipetting of entirely standards samples and
controls are done within 3 minutes. All samples, standards and control have to be run in replica
simultaneously so that every situations of trial are similar. Finally, it is commended that wells be
read in 15 minutes followed by adding of stop solution (Kucharski and Niedzielski 2013). All
chemicals must be allowed to attain room temperature prior to use and all reagents must be
mixed by moderate swirling or inversion before use to avoid turbidity or foaming. Finally, trials
with projected figures larger than 300mlu/mL should be diluted with zero stock or regular human
serum (1:100 initial dilutions) before assaying (Attar and Siddiqui 2013).

Indirect ELISA test 11
Conclusion
The enzyme-linked immunosorbent Assay is an extremely sensitive technique for
identifying and measuring either antibodies or antigens. It can be useful in testing a range of
infections and disorders and very frequently used as an early screening trial. Many disparities of
ELISA have been studied, the main types being the direct ELISA, the indirect, the sandwich and
competitive ELISA. The above experiment was able to show a positive test, but it was not the
final decision to offer a diagnosis. Therapeutic significances should certainly not be centered on
research laboratory outcomes alone even if the test result is in pact with the pieces; because
laboratory is only a share of the whole clinical image of a patient. Only scenarios where the
laboratory consequences are in satisfactory agreement with the general clinical image of the
patient, should healing importance result. The test outcomes itself should not ever be the solitary
determining factor for developing any healing effects. Additionally, the test outcomes are
effective if all controls are inside the stated scopes and if all other test limits are also within the
set assay specification.

Indirect ELISA test 12
References
Attar, S.M. and Siddiqui, A.M., 2013. Vitamin d deficiency in patients with systemic lupus
erythematosus. Oman medical journal, 28(1), p.42. Available from:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3562991/,[Accessed on 30 October 2018].
Bourdiec, A., Calvo, E., Rao, C.V. and Akoum, A., 2013. Transcriptome analysis reveals new
insights into the modulation of endometrial stromal cell receptive phenotype by embryo-derived
signals interleukin-1 and human chorionic gonadotropin: possible involvement in early embryo
implantation. PLoS One, 8(5), p.e64829. Available from:
https://doi.org/10.1371/journal.pone.0064829,[Accessed on 30 October 2018].
Choi, J. and Smitz, J., 2014. Luteinizing hormone and human chorionic gonadotropin: origins of
difference. Molecular and cellular endocrinology, 383(1-2), pp.203-213. ELSEVIER, Available
from: https://www.sciencedirect.com/science/article/pii/S0303720713005248,[Accessed on 30
October 2018].
Ezcurra, D. and Humaidan, P., 2014. A review of luteinising hormone and human chorionic
gonadotropin when used in assisted reproductive technology. Reproductive Biology and
Endocrinology, 12(1), p.95. Available from: https://doi.org/10.1186/1477-7827-12-95,
[Accessed on 30 October 2018].
Gan, S.D. and Patel, K.R., 2013. Enzyme immunoassay and enzyme-linked immunosorbent
assay. J Invest Dermatol, 133(9), p.e12. Available from:
https://www.researchgate.net/profile/Kruti_Patel4/publication/255958640_Enzyme_Immunoassa

Indirect ELISA test 13
y_and_Enzyme-Linked_Immunosorbent_Assay/links/56cc7efa08aee3cee5437a2a/Enzyme-
Immunoassay-and-Enzyme-Linked-Immunosorbent-Assay.pdf,[Accessed on 30 October 2018].
Huang, X., Xu, J., Wang, Y., Guo, C., Chen, L., Gu, X., Lai, W., Peng, X. and Yang, G., 2016.
GP50 as a promising early diagnostic antigen for Taenia multiceps infection in goats by indirect
ELISA. Parasites & vectors, 9(1), p.618. Available from:
https://parasitesandvectors.biomedcentral.com/articles/10.1186/s13071-016-1915-5,[Accessed
on 30 October 2018].
Kitahara, Y., Nakamura, K., Kogure, K. and Minegishi, T., 2013. Role of microRNA-136-3p on
the expression of luteinizing hormone-human chorionic gonadotropin receptor mRNA in rat
ovaries. Biology of reproduction, 89(5), pp.114-1. Available from:
https://doi.org/10.1095/biolreprod.113.109207,[Accessed on 30 October 2018].
Křišt’an, J., Alavi, S.M.H., Stejskal, V. and Policar, T., 2013. Hormonal induction of ovulation
in pikeperch (Sander lucioperca L.) using human chorionic gonadotropin (hCG) and mammalian
GnRH analogue. Aquaculture International, 21(4), pp.811-818. ASpringer, Available from:
https://link.springer.com/article/10.1007/s10499-012-9572-y,[Accessed on 30 October 2018].
Kuang, H., Wang, W., Xu, L., Ma, W., Liu, L., Wang, L. and Xu, C., 2013. Monoclonal
antibody-based sandwich ELISA for the detection of staphylococcal enterotoxin A. International
journal of environmental research and public health, 10(4), pp.1598-1608. Available from:
https://www.mdpi.com/1660-4601/10/4/1598/htm,[Accessed on 30 October 2018].
Kucharski, P. and Niedzielski, J., 2013. Neoadjuvant human Chorionic Gonadotropin (hCG)
therapy may improve the position of undescended testis: a preliminary report. Central European

Indirect ELISA test 14
journal of urology, 66(2), p.224. Available from:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3936151/,[Accessed on 30 October 2018].
Okda, F., Liu, X., Singrey, A., Clement, T., Nelson, J., Christopher-Hennings, J., Nelson, E.A.
and Lawson, S., 2015. Development of an indirect ELISA, blocking ELISA, fluorescent
microsphere immunoassay and fluorescent focus neutralization assay for serologic evaluation of
exposure to North American strains of Porcine Epidemic Diarrhea Virus. BMC veterinary
research, 11(1), p.180. Available from:
https://bmcvetres.biomedcentral.com/articles/10.1186/s12917-015-0500-z,[Accessed on 30
October 2018].
Salvati, E., Re, F., Sesana, S., Cambianica, I., Sancini, G., Masserini, M. and Gregori, M., 2013.
Liposomes functionalized to overcome the blood–brain barrier and to target amyloid-β peptide:
the chemical design affects the permeability across an in vitro model. International journal of
nanomedicine, 8, p.1749. Available from:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3652512/,[Accessed on 30 October 2018].
Santibañez, Á., García, J., Pashkova, O., Colín, O., Castellanos, G., Sánchez, A.P. and De la
Jara, J.F., 2014. Effect of intrauterine injection of human chorionic gonadotropin before embryo
transfer on clinical pregnancy rates from in vitro fertilisation cycles: a prospective
study. Reproductive Biology and Endocrinology, 12(1), p.9. Reproductive Biology and
Endocrinology201412:9 Available from: https://doi.org/10.1186/1477-7827-12-9, [Accessed on
30 October 2018].

Indirect ELISA test 15
Song, F., Sun, X., Wang, X., Nai, Y. and Liu, Z., 2014. Early diagnosis of tuberculous
meningitis by an indirect ELISA protocol based on the detection of the antigen ESAT-6 in
cerebrospinal fluid. Irish journal of medical science, 183(1), pp.85-88. Available from:
https://link.springer.com/article/10.1007/s11845-013-0980-4, [Accessed on 30 October 2018].
Wan, X., Wang, W., Liu, J. and Tong, T., 2014. Estimating the sample mean and standard
deviation from the sample size, median, range and/or interquartile range. BMC medical research
methodology, 14(1), p.135. Available from: https://doi.org/10.1186/1471-2288-14-135,
[Accessed on 30 October 2018].
Youssef, M.A., Van der Veen, F., Al-Inany, H.G., Mochtar, M.H., Griesinger, G., Nagi
Mohesen, M., Aboulfoutouh, I. and van Wely, M., 2014. Gonadotropin-releasing hormone
agonist versus HCG for oocyte triggering in antagonist-assisted reproductive
technology. Cochrane Database Syst Rev, 10(10). Available from:
https://pure.uva.nl/ws/files/2771809/178085_05_1_.pdf, [Accessed on 30 October 2018].
Zarei, A., Parsanezhad, M.E., Younesi, M., Alborzi, S., Zolghadri, J., Samsami, A., Amooee, S.
and Aramesh, S., 2014. Intrauterine administration of recombinant human chorionic
gonadotropin before embryo transfer on outcome of in vitro fertilization/intracytoplasmic sperm
injection: A randomized clinical trial. Iranian journal of reproductive medicine, 12(1), p.1.
Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4009584/, [Accessed on 30
October 2018].

Indirect ELISA test 16
Reflections
To plan the research, we had to make some big questions. First, what type of ELISA did
we want to run? There are many types of Elisa, but we narrowed it down to indirect ELISA.
Selection of antibodies is important step as every antibody as its own implications. I was
involved in washing plates after every development phase which was essential in ELISA to avert
non-specific interfaces. I was directed to wash the plates by TBS or PBS after every antibody
growth stages. In the detection and growth stage, we had to use the conjugated antibody. The
enzyme produced colour when exposed to one of its substrates such as tetramethylbenzidine.
Finally, I was able to stop the reaction using 2M sulphuric acid which converted the substrates
colour. In the reading phase, I was able to read the intensity of colour signal on
spectrophotometer set to a 450nm wavelength that is suitable for the substrate used. I had an
opportunity of using automatic plate reader set up to read the entire 96 well plates in one reading,
storing the info in an orderly and planned way. This study was selected to shows that ELISA is a
popular assay that uses antibodies and colour alterations to detect peptides, proteins, antibodies
or biomolecules in sophisticated blends. They are popular because of their reliability, specificity
and easy to use, and can easily be scaled up to process multiple samples concurrently.
Personally, the study has enabled me to know the advantages and disadvantages of each step of
the ELISA tests. It has given me courage and motivation to perform the remaining types of
ELISA in the analysis of various sample specimens under minimal supervision. In the corporate
world, the study as enlightened me on importance of efficacy, precision, versatility, sensitivity,
and flexibility of the process in my near future place operation. The method will assist me in
future as I plan to involve myself in the stem cell research.

Indirect ELISA test 17