Recombinant Human Erythropoietin Quantification using Enzyme-Linked

Immunosorbent Assay (ELISA)

Bella Anbar Fauziyyah1, David Alberto Christian2

1,2
Biomanufacturing Training Program, Indonesia International Institute for Life Science (i3L)
1
bella.anbar@btp.i3l.ac.id, 2david.albertho@btp.i3l.ac.id

1. Introduction
1.1 Description about EPO and ELISA
Erythropoietin (EPO) is an endogenous hormone of glicoproteic nature secreted by the kidneys
and synthesized particularly in specific epithelial cells that line renal peritubular capillaries [1]. It is also
produced in perisinusoidal cells in the liver. EPO is the main regulator of the erythropoiesis [2]. Human
EPO was first isolated and later purified from urine in the 1970s. Interest in developing clinical uses for
EPO led to the discovery of the gene encoding EPO, and several groups devised recombinant DNA
methods to produce EPO by the mid-1980s. Availability of this compound in the market dramatically
increased in the 80s, with the advent of recombinant DNA technology. EPO synthetic form, recombinant
human EPO (rhEPO), started to be marketed in 1983. The administration of recombinant human
erythropoietin (rHuEPO) and its analogues provides enormous benefit in the prevention and reversal of
anaemia in chronic kidney disease (CKD), malignancy and AIDS, supports autologous blood collection
and improving quality of life of patients in constant need of blood transfusions, or with anemia due to
chronic renal failure. rhEPO-type substances represent the largest market of a class of
biopharmaceuticals, with global estimated sales of 1010 euro per annum [3]. Chinese hamster ovary
(CHO) cells are most commonly used for the large-scale pharmaceutical manufacture
of erythropoietin and the quantitation of erythropoietin level in both serum and during manufacturing
plays important role in pharmaceutical manufacture of erythropoietin.
The enzyme-linked immunosorbent assay (ELISA) a biochemical assay that uses antibodies
and an enzyme-mediated color change to detect the presence of either antigen (proteins,
peptides,hormones, etc.) or antibody in a given sample. ELISA widely used as diagnostic tools in
medicine and as quality control measures in various industries for example for the detection and
quantification of specific protein existed in the sample. ELISA also used as analytical tools in biomedical
research for the detection and quantification of specific antigens or antibodies in a given sample. In this
laboratory practice, the concentration of recombinant human Erythropoietin (rhEPO) was measured
using ELISA.

1.2 Objective

Determine the concentration of of recombinant human Erythropoietin (rhEPO) using ELISA.

2. Material & Method

2.1 Material

The material used in this laboratory practice is comprised of eBioscience Microwell Plate coated
with monoclonal antibody to human Erythropoietin, Biotin-Conjugate monoclonal anti-Erythropoietin
antibody, sample diluent, Streptavidin-HRP , 100 mIU/ml Erythropoietin Standard lyophilized after
reconstitution, wash buffer concentrate (PBS with 1 % Tween 20), assay buffer concentrate (PBS with
1% Tween 20, 10% BSA), Substrate Solution (tetramethyl-benzidine), Stop Solution (1M Phosphoric
acid), Nikon Eclipse TE300 inverted microscope, Vortex Genie 2, Hybrid Capture System Digene
shaker, Biologix 200,1000L pipet tips, Assistent haemocytometer, Rainin pipet gun, Eppendorf
Research plus micropipet 100 1000L , 20 - 200L, 10 - 100L, Eppendorf thermomixer, Falcon
tissue culture plate 6 well, Tecan NanoQuant infinite M200 and Adhesive film.
2.2 Method

The Method used in this practice refers to Human EPO Coated ELISA Kit: Product Information
and Manual BMS2035 or BMS2035TEN using 3 strips of microwell plate each assay.

2.2.1 Wash Buffer Preparation

Wash buffer was reconstituted by twenty fold dilution (1:20) with water type I from its
concentrated stock. The wash buffer were stored at 4oC.

2.2.2 Assay Buffer Preparation

Wash buffer was reconstituted by twenty fold dilution (1:20) with water type I from its
concentrated stock. The assay buffer were stored at 4oC.

2.2.3 Biotin-conjugated

Biotin-conjugated was reconstituted by one hundred fold dilution (1:100) with assay buffer from
its concentrated stock. The biotin conjugated should be used within 30 minutes after dilution.

2.2.4 Streptavidin HRP

Streptavidin - HRP was reconstituted by one hundred fold dilution (1:100) with assay buffer
from its concentrated stock. The streptavidin HRP should be used within 30 minutes after dilution.

2.2.5 Standard Preparation

Reconstitute human Erythropoietin standard by addition of distilled water until the concentration
of reconstituted standard reach 100 mIU/ml. Mix gently to insure complete and homogeneous
solubilization

2.2.6 Standard Dilution

100 l of Standard stock (100 mIU/ml) were poured in first well plate containing 100 l sample
diluent , then re-suspend it. Then, transfer 100 l solution of first well to second well until 7 th well. This
protocol was done for twice.

2.2.7 Control Preparation

Reconstitute by adding 250 l distilled water to lyophilized controls. Mix gently to ensure
complete and homogeneous solubilization.

2.2.8 Seeding and Spiking HeLa Cells with rhEPO

Cultured HeLa cells in T25 flask were trypsinized. After trypsinized, the HeLa cells were seeded
to well plate in such a way that each well contains 0.3 x 106 cells at the least. After that, The HeLa cells
were incubated overnight, it was spiked with unknown concentration of sample rhEPO. The samples
were then incubated for 30 minutes until 1 hour.

2.2.9 ELISA Test Protocol

Microwell strips were washed twice with 400 l Wash Buffer per well. Then let the Wash Buffer
to sit in the wells for about 15 seconds before aspiration. 100 l of Sample Diluent were added to
standards and blank wells, and 50 l to sample and control wells. 50 l of each sample was added in
duplicate to the sample wells and 50 L of control were added to the control wells. The well configuration
is shown by table 1.
 Table 1. Arrangement of Standards, Sample, Blank and Control in Well
1 2 3

A Standard 1 (50 mIU/ml) Standard 1 (50 mIU/ml) Sample 1a

B Standard 2 (25 mIU/ml) Standard 2 (25 mIU/ml) Sample 1b

C Standard 3 (12.5 mIU/ml) Standard 3 (12.5 mIU/ml) Sample 2a

D Standard 4 (6.25 mIU/ml) Standard 4 (6.25 mIU/ml) Sample 2b

E Standard 5 (3.125 mIU/ml) Standard 5 (3.125 mIU/ml) Control High

F Standard 6 (1.5625 mIU/ml) Standard 6 (1.5625 mIU/ml) Control High

G Standard 7 (0.78125 mIU/ml) Standard 7 (0.78125 mIU/ml) Control Low

H Blank Blank Control Low

50 l of Biotin-Conjugate was added to all wells, cover with an adhesive film and incubate at room
temperature for 1 hour with agitation on a microplate shaker set at 400 rpm. Adhesive film was removed
and all the liqiud in the wells were discarded. Then all wells washed 6 times with 400 l wash buffer.
Then, 100 l of diluted Streptavidin-HRP was added to all wells. All wells then was covered with an
adhesive film and incubated at room temperature for 15 minutes with agitation on a microplate shaker
set at 400 rpm. The adhesive film was removed and then all liquid in the wells were discarded. Then all
wells washed 6 times with 400 l wash buffer. After that, 100 l of TMB Substrate Solution was added
to all wells. The microwell strips then were incubated the at room temperature for about 10 - 15 min.
Avoid direct exposure to the light. After the highest standard has developed a dark blue color, the
absorbance was checked by spectro-photometer at 620 nm, when the highest standard reach 0.9
absorbance, stop solution was added. Absorbance of each microwell was read on a spectro-photometer
at 450 nm.

3. Result
3.1 Data

Figure 1. Absorbance of Standard, Blank, Control Low, Control High and Sample
rhEPO Before Addition of Stop Solution ( = 620 nm, number of flashes = 3)
Figure 2. Absorbance of Standard, Blank, Control Low, Control High and Sample of
rhEPO After Addition of Stop Solution ( = 450 nm, number of flashes = 3)

Figure 3. ELISA Plate that Contain Standard, Blank, Control Low, Control High and
Sample of rhEPO Before Addition of Stop Solution

Figure 4. ELISA Plate that Contain Standard, Blank, Control Low, Control High and
Sample of rhEPO After Addition of Stop Solution
3.2 Data Analysis

Table 2. Absorbance of rhEPO Standard and Blank

Absorbance
EPO
Standard 1 Standard 2 Standard
Concentration Standard Standard Standard
(Minus (Minus Average (Minus
(mIU/mL) 1 2 Average
Blank 1) Blank 2) Blank Average)
50 2,8742 2,8464 2,8603 2,7832 2,7613 2,77225
25 1,2612 1,3613 1,31125 1,1702 1,2762 1,2232
12,5 0,5799 0,6641 0,622 0,4889 0,5790 0,53395
6,25 0,2687 0,3568 0,31275 0,1777 0,2717 0,2247
3,125 0,2046 0,2236 0,2141 0,1136 0,1385 0,12605
1,5625 0,1595 0,116 0,13775 0,0685 0,0309 0,0497
0,78125 0,102 0,0959 0,09895 0,0110 0,0108 0,0109
Blank 0,091 0,0851
Blank average 0,08805

rhEPO Standard Curve (Average)

3

2,5

2
Absorbance

1,5
y = 0,0559x - 0,0866
1 R = 0,9957

0,5

0
0 10 20 30 40 50 60
rhEPO Concentration (mIU/mL)

Figure 5. rhEPO Standard Curve (Average)

rhEPO Standard Curve (Standard 1)

3,0

2,5
Absorbance

2,0
y = 0,056x - 0,1061
1,5
R = 0,9912
1,0

0,5

0,0
0 10 20 30 40 50 60
rhEPO Concentration (mIU/mL)

Figure 6. rhEPO Standard Curve (Standard 1)

 rhEPO Standard Curve (Standard 2)
3,0

2,5

Absorbance 2,0
y = 0,0558x - 0,0672
1,5
R = 0,9985
1,0

0,5

0,0
0 10 20 30 40 50 60

rhEPO Concentration (mIU/mL)

Figure 7. rhEPO Standard Curve (Standard 2)

Table 3. Back Calculated Concentration of Calibration Standards

Back Calculated Concentration Limits

Concentration Concentration Concentration Using -20% (25% +20% (25%
Using Standard Using Standard Standard Average except for LLOQ except for LLOQ
Curve 1 (mIU/mL) Curve 2 (mIU/mL) (mIU/mL) and ULOQ) AND ULOQ)
51,59464286 50,68996416 51,14221825 40 60
22,79107143 24,07526882 23,43112701 20 30
10,625 11,58064516 11,10107335 10 15
5,067857143 6,073476703 5,568872987 4,6875 7,8125
3,923214286 3,686379928 3,80411449 2,34375 3,90625
3,117857143 1,758064516 2,438282648 1,171875 1,953125
2,091071429 1,397849462 1,744186047 0,5859375 0,9765625

Table 4. Absorbance and Concentration of rhEPO Control and Sample

Concentration
Concentration Concentration
Sample (Standard
(Standard (Standard
Sample Absorbance (Minus Curve
Curve 1) Curve 2)
Blank) Average)
(mIU/mL) (mIU/mL)
(mIU/mL)
Control High 1 1,5485 1,46045 55,94821429 54,84670232 55,35062612
Control High 2 1,8408 1,75275 66,3875 65,26737968 65,80858676
Control Low 1 0,2511 0,16305 9,6125 8,593582888 8,932021467
Control Low 2 0,3041 0,21605 11,50535714 10,48306595 10,82826476
Sample 1a 0,5003 0,41225 18,5125 17,47771836 17,84794275
Sample 1b 0,4319 0,34385 16,06964286 15,03921569 15,40071556
Sample 2a 0,4291 0,34105 15,96964286 14,93939394 15,30053667
Sample 2b 0,4037 0,31565 15,0625 14,03386809 14,39177102
 Table 5. Coefficient of Variance of rhEPO Sample 1a and 1b

Concentration
Concentration Concentration
(Standard Curve
Sample (Standard Curve (Standard Curve 2)
Average)
1) (mIU/mL) (mIU/mL)
(mIU/mL)
Sample 1a 18,5125 17,47771836 17,84794275
Sample 1b 16,06964286 15,03921569 15,40071556
Standard
1,727360851 1,724281777 1,730450942
Deviation
Mean 17,29107143 16,25846702 16,62432916
Coefficient of
0,099899006 0,106054388 0,104091475
Variance (CV)
CV (%) 9,989900616 10,60543884 10,40914749

Table 6. Coefficient of Variance of rhEPO Sample 2a and 2b

Concentration
Concentration Concentration
(Standard Curve
Sample (Standard Curve (Standard Curve 2)
Average)
1) (mIU/mL) (mIU/mL)
(mIU/mL)
Sample 2a 15,96964286 14,93939394 15,30053667
Sample 2b 15,0625 14,03386809 14,39177102
Standard
0,641446866 0,640303467 0,642594356
Deviation
Mean 15,51607143 14,48663102 14,84615385
Coefficient of
0,041340804 0,044199612 0,043283558
Variance (CV)
CV (%) 4,134080387 4,419961177 4,328355764

Table 7. Coefficient of Variance of rhEPO Control High 1 and 2

Concentration
Concentration Concentration
(Standard Curve
Sample (Standard Curve (Standard Curve 2)
Average)
1) (mIU/mL) (mIU/mL)
(mIU/mL)
Control High 1 55,94821429 54,84670232 55,35062612
Control High 2 66,3875 65,26737968 65,80858676
Standard
7,381689719 7,368531627 7,394894889
Deviation
Mean 61,16785714 60,057041 60,57960644
Coefficient of
0,120679227 0,122692219 0,122069048
Variance (CV)
CV (%) 12,0679227 12,2692219 12,2069048
 Table 8. Coefficient of Variance of rhEPO Control Low 1 and 2

Concentration
Concentration Concentration
(Standard Curve
Sample (Standard Curve (Standard Curve 2)
Average)
1) (mIU/mL) (mIU/mL)
(mIU/mL)
Control Low 1 9,6125 8,593582888 8,932021467
Control Low 2 11,50535714 10,48306595 10,82826476
Standard
1,338452122 1,336066289 1,34084649
Deviation
Mean 10,55892857 9,538324421 9,880143113
Coefficient of
0,126760221 0,140073479 0,135711242
Variance (CV)
CV (%) 12,67602212 14,00734794 13,57112417

4. Discussion
4.1 rhEPO Standard Calibration Curve

4.1.1 Coefficient Determination of rhEPO Standards Calibration Curve

Based on the results of the absorbance and calculation of the absorbance of rhEPO standards
minus blank in Table 2, there was obtained 3 standard curve that consist of rhEPO standard curve
average (Figure ), rhEPO standar curve 1 (Figure), and rhEPO standard 2 (Figure). Standard curve
average generated the equation: y = 0,0559x - 0,0866 with the R2 (coefficient of determination) value
was 0,9957.From its coefficient determination value, it means that 99,57% of the variation in the
dependent variable (y or absorbance) can be explained by the independent variable ( x or
concentration). It also means that 99,57% of the point on the regression line fits the data which means
that the regression model fits the data well and 99,57% accurately predict the future outcomes.
Meanwhile, rhEPO standard curve 1 generated the equation: y = 0,056x - 0,1061with the R2 (coefficient
of determination) value was 0,9912.From its coefficient determination value, it means that 99,12% of
the variation in the dependent variable (y or absorbance) can be explained by the independent variable
( x or concentration). It also means that 99,12% of the point on the regression line fits the data which
means that the regression model fits the data well and 99,12% accurately predict the future outcomes.
The regression model rhEPO standard curve 2 fits the data well. rhEPO standard curve 2 has the
equation : 0,0558x - 0,0672 with the R2 (coefficient of determination) value was 0,9985. It means that
99,85% of the variation in the dependent variable (y or absorbance) can be explained by the
independent variable (x or concentration). It also means that 99,85% of the point on the regression line
fits the data and 99,85% accurately predict the future outcomes.

4.1.2 Back Calculated of rhEPO Standards Calibration Curve

Refers to Table 3 about back calculated concentration of calibration standards, all the rhEPO
standards calibration curve (standard curve average, standard curve 1, standard curve 2) comply the
criteria from EMEA Guideline on bioanalytical method validation although Standard Curve 1 and
Standard curve average has 2 concentration that out from the limit, and Standard Curve 2 has 1
concentration that out from the limit. The back calculated concentrations of the calibration standards
should be within 20% of the nominal value, except for the LLOQ for which it should be within 25%
and at least 75% of the calibration standards, with a minimum of six, and all the rhEPO standards
calibration curve (standard curve average, standard curve 1, standard curve 2) comply this criteria and
all calibration standard curves should be accepted.
4.2 Control Low and High

Refers to table 4, all controls are in the range of concentration of control that should be. From
the table 4, the concentration of control low 1 and 2 were 9.6125 mIU/mL, 8.5935 mUI/mL, and 8,93202
mIU/mL, 11.5053 mIU/mL, 10.4830 mIU/mL, and 10.828 mIU/mL which means that these concentration
correspond to the concentration that should be (5-20 mIU/mL). For the control high, from the table 4 the
concentration of control high 1 and 2 were 55.948 mIU/mL, 54.846 mIU/mL, 55.350 mIU/mL, 66.3875
mIU/mL, 65.267 mIU/mL, and 65.808 mIU/mL. These concentration were also correspond to the
concentration that should be (50 150 mIU/mL). For the precision of the analytical method, the
coefficient of variance (CV) was calculated for the control low and control high. The precision of the
analytical method describes the closeness of repeated individual measures of analyte. The CV value
should not exceed 20%. Refers to table 8, %CV of Control low were 12.676%, 14.007%, and 13.571%.
Meanwhile, refers to table 7, %CV of Control high were 12.067%, 12.269%, and 12.206%. These %CV
results from both control comply the criteria that CV value should not exceed 20% which means that
this result has a good precision.

4.3 Sample

There were two sample that used in this laboratory pratice, sample 1 and 2 and each sample
was duplicated thus there were 4 sample in this laboratory practice (sample 1a, sample 1b, sample 2a,
and sample 2b). The concentration of all sample was 10 mIU/mL. Refers to table 4, all sample
concentration exceed the actual concentration (10 mIU/mL). This things happen due several things
such as interference factors in immunoassay such as sample carryover due to inadequate assay
washing or failure to detect a sample clot, samples or assay reagents contaminated with substances
that interfere with measurement of the label like enzyme inhibitors, fluorophores. For the precision of
the analytical method, the coefficient of variance (CV) was calculated for the sample 1a, 1b and sample
2a, 2b. The precision of the analytical method describes the closeness of repeated individual measures
of analyte. The CV value should not exceed 20%. Refers to table 5 and 6, %CV of sample 1a, 1b, 2a,
and 2b were not exceed 20%. These %CV results from all samples shows that this result has a good
precision. This result does not same with the accuracy of the analytical method. The criteria for good
accuracy is the value of percent of recovery does not exceed or lower than 20% from the actual
concentration and the result from all samples show that it exceed and lower than 20% from the actual
concentration.

5.Conclusion
Standard curve average generated the equation: y = 0,0559x - 0,0866 with the R2 value was
0,9957. rhEPO standard curve 1 generated the equation: y = 0,056x - 0,1061with the R2 value was
0,9912. rhEPO standard curve 2 has the equation : 0,0558x - 0,0672 with the R2 value was 0,9985. The
concentration of sample 1a were 18.512 mIU/mL, 17.477 mIU/mL, and 17.8479 mIU/mL. Concentration
of sample 1b were 16.069 mIU/mL, 15.039 mIU/mL, 15.400 miu/ml. Meanwhile concentration of sample
2a were 15.969 mIU/mL, 14.939 mIU/mL, 15.300 mIU/mL. Concentration of sample 2b were 15.0625
mIU/mL.14.033 mIU/mL, and 14.391 mIU/mL. All samples and controls shows a good precision with all
%CV value not exceed 20%.

6. Reccomendation
There are some evaluation to get the better result of ELISA. It consist of minimizing the
interference in immunoassay, full validation of bioanalytical method, improve pipetting technique to
have more reliable and accurate data, work with speed and accuracy.

7. References
[1] Adamson JW, Regulation of red blood cell production, Am J Med, 101, (1996), pp.4-6.
[2] Rafael Maia de Almeida Bento; Lcia Menezes Pinto Damasceno; Francisco Radler de Aquino
Neto, Recombinant human erythropoietin in sports: a review. Rev Bras Med Esporte, vol.9, no.3,
(2003), pp.181-190.
[3] Wolfgang Jelkmann, Recombinant EPO productionpoints the nephrologist should know,
Nephrol Dial Transplant, vol.22 no.10, (2007), pp. 2749-2753.