Developing competitive ELISA

Developing competitive ELISA

1-300 Abstract
2-introduction need to be paraphrased
3-methods need to be change the style of writing to scientific English in a style how methods of experiment is written
4- discussion you should write 2500 with 30 reference to support the interpretation of the graph
5-conclusion should be 200
6- references should be in Vancouver style

                                                                DECLARATION

• This final project report does not contain any material which has been accepted for theaward of any other degree or diploma and to the best of my knowledge and belief,contains no material written or published previously by another person, except wheredue reference is made in the text.

• Signed:
• Date: 19 October 2014

Table of Contents

List of figures……………………………………………………………………..……………………4

List of Tables…………………………………………………………………………………………..5

List of Abbreviations…………………………………………………………………………………..6

Abstract…………………………………………………………………………………………………7

Chapter 1: Introduction……….…..…………………………………………………………………….8

Chapter 2: Materials and Methods ……………………………………………………………………9

Chapter 3: Results…………………………………………………………………………………….15

Chapter 4: Discussion……………..…………………………………………………………………..25

Chapter 5: Conclusion…………………………………………………………………………………29

Acknowledgment……………………………………………………………………………………….30

References………………………………………………………………………………………………30

Appendix……………………………………………………………………………………………….34

List of Figures

Figure 1 : Selection of extraction solvent……………………………………………………………16

Figure 2:  Calibration curve………………………………………………………………………….17

Figure 3:Levey Jennings chart: ……………………………………………………………………..19

Figure 4:Precision profile …………………………………………………………………………….21

Figure 5: Precision profile (plot 2)…………………………………………………………………….31

Figure 6: Linearity …………………………………..………………………………………………..24

Figure 7:Passing Bablok……………………………………………………………………………..24

Figure 8:Difference Plot………………………………………………………………………………26

Figure 9: Sigma Metrics Chart………………………………………………………………………..28 

List of Tables

Table 1: Materials and manufacturers …………………………………………………………….9

Table 2:standards concentration and the volume……………………………..…………………10

Table 3: Preparation of the five mixtures for linearity ……………………………………….….14

Table 4:QC concentration from the calibration curve……………………………………………17

Table 5:Impression result for high value and low values……………………………………..…..18

Table 6:Concentration of LOB, LOD, high and low values………………………………………20

Table 7:Assigned and assayed concentration for the linearity plot……………………………….22

Table 8:sigma Metrics Chart………………………………………………………………………24

List of Abbreviations

ELISA	Enzyme-Linked Immunosorbent Assay
CV%	The percentage of Coefficient of variation
RCPA QAP	Quality Assurance Programs
LoB	Limit of Blank
LoD	Limit of Detection
LoQ	Limit of limit of quantitation
LC–MS/MS	Liquid chromatography–mass spectrometry
GC-MS	Gas chromatography–mass spectrometry
BSA	Bovine serum albumin

Abstract

ELISA is undoubtedly one of the extensively utilized biochemical techniques hence considered a routine procedure in most clinical laboratories. A combination of ELISA with other throughput technologies has significantly revolutionized the way laboratory procedures are conducted particularly those involved in purification of assays or analytes. Through development and validation of a competitive ELISA assay to measure levels of serum cortisol making comparison of the results obtained from ELISA with those obtained from the LCMS/MS, it is possible to conduct an evaluation of the highly reproducible, most reliable, highly accurate and sensitive method for the quantification of cortisol between the methods that are considered. Moreover, considering that there was need in this laboratory project to extract cortisol, then it can be highly justified that the laboratory project would definitely begin with the development of the technique to extract the cortisol which began by choosing the solvent to be used for the extraction of cortisol whereby ethyl acetate, hexane, and MTBE were considered and eventually the extracts were run on LCMS/MS. The project validated the techniques by evaluating the accuracy, sensitivity, linearity and imprecision for the ELISA. However, the linearity plot results indicated that that there was consistency between the linearity plot results with those of the calibration curve where the cortisol’s concentration in the upper limit was 750nmol/L. Thus, this implies that  in spite of the ELISA’s performance  been not very good for the considered analyte, a significant correlation was in existence upon comparing between the two methods.

Keywords:

Cortisol, development, validation, immunoassay, mass spectrometry assay, ELISA, LC-MS, LoB, LoD, LoQ

CHAPTER 1: INTRODUCTION

Enzyme-Linked Immunosorbent Assay (ELISA) is undoubtedly one of the most used biochemical techniques hence considered a routine procedure in most research and clinical laboratories¹. This is mainly because ELISA is a detection method that exploits the ability of antibodies to bind specifically and very tightly to a particular compound such as the antigens. The detection of the antibodies is done through a secondary antibody that is linked to a quantification or visualization strategy². As a result ELISA has a wide usage in the diagnosis diseases and screening for the presence of some drugs in the body³. LC-MS/MS Chromatography is a method of separating components in a mixture based on the differences in partitioning behavior between a stationary phase and a flowing mobile phase². Moreover, ELISA is usually combined with other biochemical analysis techniques which include liquid chromatography tandem mass spectrometry (LC–MS/MS), has led to major breakthroughs in quantitative bioanalysis in biomedical sciences mainly because of the inherent sensitivity, specificity, and speed⁴. According to Lequin⁵ due to the above mentioned characteristics which are inherent in LC-MS/MS, the technique has received general acceptance as the preferred technique for the quantification of small molecule metabolites, drugs, as well as other xenobiotic biomolecules in biological matrices. Techniques that are liquid chromatography tandem mass spectrometry (LC-MS/MS) based have gained wide usage nowadays in the analysis of steroid serum. For instance, in the detection of serum aldosterone liquid chromatography–mass spectrometry (LC–MS) method is usually regarded an upper method¹. However, LC–MS/MS technique should be used as a reference method because it mainly offers numerous advantages compared to GC-MS⁵.The aim of the experiment was to develop a competitive ELSA for serum cortisol to be acceptable standard essay and to validate Elisa assay by comparing with LC-MS/MS reference method.

CHAPTER 2: MATERIALS AND METHODS

2.1 Materials and Reagents

For sample extraction, Methanol and Ethyl acetate and Monoclonal cortisol antibody (5.4mg/mL) was used for labeling antigen.Tween20, Bovine serum albumin( BSA), Cortisol-1,2-d2 internal standard and Lumigen PS-atto (substrate)were used in this competitive ELISA assay and the rest of the materials used and their manufactures are provided in the appendix. For the purpose of diluting pure cortisol with 1000nmol/L Sercon was used. Cortisol Quality Controls samples were used with 3 different levels for testing the experiment reliability and acceptability. Cortisol sample was used by RCPA QAP general chemistry program and Endocrine program. The main equipment used in this experiment are Fume hood, Eppendorf centrifuge, vortex equipment for sample preparation.

Table 1: showing the all materials used and their manufacturers.

	Materials	(manufacturer name, city, state)
1	Bovine serum albumin	Sigma-Aldrich, 3050 Spruee St ST. Luis, M063103 USA
2	Cortisol-1,2-d2 internal standard	CIDIN IsotopcpInc, 88 Leacock St Pointe-Claire, Quebec  Canada
3	Cortisol Quality Controls	Bio-Rad Laboratories, Irvine,  CA
4	Cortisol-3- Carboxymethoxylamine-(HRP)	MyBiosource, San Diego, California, USA
5	ELISA 96 well plate	NVNC A/S, kamstrvpvej, Roskilde Denmark
6	ELISA reader	Perkin Elmer. Inc, Waltham, Massachusetts
7	Ethyl acetate	Merck KGaA, Darmstadt, Germany
8	Endocrine calibrators (cortisol)-RCPA	Thermo Scientific, Rockford IL USA
	Eppendolf centrifuge 5424, Eppendorf AG 22331	Haburg Germany
9	Hexane	Merck KGaA, Darmstadt, Germany
10	Hydrochloric acid (1M)	Merck KGaA, Darmstadt, Germany
11	Hydrated Disodium Hydrogen Phosphate (Na2HPO4.12H2O)	Ajax Finechem, seven hills. NSW
12	Lumigen PS-atto (substrate)	Lumigen, Southfield, Michigan
13	Liquid chromatography-tandem mass spectrometer (LC-MSMS)	Agilent Technologies, Santa Clara, California
14	LC –ms/ms  Agilent Technologies, ms/ms=6490, LC=model 1290
15	Methanol	Merck KGaA, Darmstadt, Germany
16	Methyl-Tert-butyl ether (MTBE)	Sigma Aldrich G, St. Louis, Missouri
17	Monoclonal Cortisol Antibody ( 5.4mg/mL)	MyBiosource, Irvine, California
18	Potassium Chloride (KCL)	Clinical Biochemistry lab, Rmit, Bundorra
19	Potassium dihydrogen phosphate ( KH2PO4)	Ajax Scientific, Carlsbad CA
20	Pure cortisol	Steraloids Inc., Newport, Rhode Island
21	Sodium hydroxide (1M)	Merck, DarmStadt Germany
22	Sulphuric acid	Merck, DarmStadt Germany
23	Sodium Hydrogen carbonate	Sigma-Aldrich, St Luis  USA
24	Sodium Hydrogen phosphate	Sigma-Aldrich, St Luis  USA
25	Sodium Chloride	Merck, DarmStadt Germany
26	Sodium Bicarbonate ( Na2CO3)	Clinical Biochemistry lab, Rmit, Bundorra
27	Seracon ( matrix matched cortisol free serum)	Sigma-Aldrich, St Luis  USA
28	Sodium carbonate ( Na2CO3)	Merck, DarmStadt Germany
29	Sodium Chloride (NaCl)	Sigma-Aldrich, St Louis  USA
30	RCPA chemical pathology QAP samples	Aalto Scientific, Carlsbad, CA
31	Rotek Instruments PTY ltd	Buronia VIC 3155
32	Tetra-Methy-Benzidine (3,3’,5,5’-TMB)	Thermo Scientific, Rockford IL USA
33	Tween 20	Sigma-Aldrich, St Louis  USA
34	Thermo Scientific, multiskan spectrum
35	Thermofisher Scientific	Caribbean DV Scoreshy VIC 3179

2.2 Buffers

The main buffer used in this experiments were phosphate buffer saline (PBS), washing, coating and blocking buffer. Coating buffer was crucial for efficient immobilization. The pH was adjusted to be 9.6 as wrong pH can affect the assay. The reagents used in Coating buffer were made of 1M Na₂CO₃ the amount taken was 3.03g, 1M of NaHCO₃the amount taken was 6.0 and 1mL of distill water. To make a 10 times concentrated phosphate buffer saline (PBS)buffer. The reagents used in 10X PBS buffer were Na₂HPO₃.12H₂O (1.16g), KH₂PO₄ (0.1g), NaCl (4.0g), KCl (0.1g), distill water (500 mL) and the pH was adjusted to be 7.4.For removing the component that are not bound, washing buffer was used. The reagents used in washing buffer were 0.05% (v/v) of Tween 20 in PBS. Blocking buffer used as a blocker of non-specific protein-surface binding. The reagents used in blocking buffer 1% BSA (Bovine serum albumin) solution phosphate buffered saline (PBS), blocking and washing buffer. All these buffers were prepared to perform ELISA assay. The details of the buffer and their reagent are show in the table above.

2.3 Liquid extraction Protocol for ELISA

About 100 µL of unknown or control samples were put in labeled microfuge tubes followed by subsequent addition of about 100 µL of methanol and pulse vortexted for 2 minutes. After that about 100 µL of distilled water were added to the tube and vortexted for 2 minutes; this was followed by subsequent addition of about 800µL of Ethyl acetate and pulse vortexted for 5 minutes. The tubes were then centrifuged for 5minutes at 8000 rpm. This was followed by the transfer of about 500µL of the supernatant from each tube to new glass tubes and dried in 37C. 

2.4 Sample Extraction Protocol for ELISA

About 100 L of the samples were added to each tube then 100 µL of methanol were added. The tubes were vortexing for 2 minutes and about 100 µL of distill water were then vortex for 2 minutes followed by addition of about 800µL of ethyl acetate then vortex for 5mins. All the tubes were then centrifuged at 8000 R.P.M for 5 minutes and about 500 µL of supernatant were transferred into clean tubes. The sample was dried at 37^oC. Finally, the sample was reconstituted with 500 µL of PBS or ceracon.

2.5ProtocolLiquid Liquid extraction for LCMSMS

About 100 µL of unknown or control samples were taken in labeled microfuge tubes. The same amount of cortisol-1.2- d₂ internal standard plus methanol solution was added and the tubes were pulse vortexted for 60 seconds. About 100 µL of distilled water were added to the tubes and vortexted again for 60 seconds followed by addition of about 500µL of ethyl acetate to the tubes and vortexted for 5 minutes. The tubes were then placed in the centrifuge and spanned at 1000 rpm for 5 minutes.  About 200 µL of the supernatant was then transferred from each tube to new glass tubes and dried down with Nitrogen. The tubes were then reconstituted with 250 µL of 70 % methanol and were then vortexted for 20 seconds.

2.6 ELISA protocol

About 100 µL of monoclonal antibody were added to each well on the plate and the plate was then covered with parafilm and incubated at 37^oC for 2 hours. The plate was blocked with blocking buffer incubated at 37 Cº for 1 hour. After that, the plate was washed 3 times with 200uL of PBST and rinsed 1 time with  200 µL of 1X PBS. Around 100 µL of extracted sample, control and standards were added to assigned wells then incubated for 15 minutes at 37Cº. About 100 µL of Cortisol-3-CMO( HRP) conjugate (1:10,000 dilution factor) followed by incubation for 1 hour at 37 Cº. The plate was washed with 200 µL of PBST 3 times followed and then rinsed once with 200 µL of 1X PBS. Lastly, 100 µL of lumigen which considered as substrate was added then followed by immediate detection of the signal³⁹.

2.7 Method development of competitive ELISA assay

Three RCPA QAP samples low, medium and high samples along with Seracon as a blank were run on ELISA as per the protocol to obtain a response curve.

For the low sample the range was 80-08 (234 nmol/L), QC1: 40661 (127 nmol/L), Range = 107-147, QC2: 40662 (427 nmol/L), Range = 359-495, QC3: 40663 (937 nmol/L), Range = 787-1087

2.7.1Pipette Calibration (Accuracy):

The 4 pipettes that were used throughout the project were calibrated. The pipettes used were the 100 – 1000 µL and 20 – 200 µL. The percentage error was calculated following this equation –

%Error = [(Average Weight – 1.000g)/1.000g] x100.

2.7.2 pH of Buffers:

The pH of all buffers and consistency was checked (visual check). For example, Coating Buffer (pH=9.6), Washing Buffer (pH=7)and  PBS (1X Buffer) (pH=7.4)

2.8 Methods Validation:

Method validation was done to ensure that the methods done in this experiment is fit for purpose after approving the effectiveness of the standard curve for the ELISA assay. The validation was performed by evaluating imprecision, linearity, sensitivity and finally the method was compared to LCMSMS. The QC materials used were from RCPA QAP.

2.8.1 Calibration Curve

Seven standards were prepared as described for calibration curve as shown in Table 1. Then all standards were extracted (Liquid-Liquid extraction with MTBE) and run on ELISA. The calibration curve was plotted using the Readerfit program.

Table 2. Seven Standards, their concentration and the final cortisol concentration

Standards	Concentration	Total Volume (μl)	Diluent Volume (µL)	Cortisol (µL)
1	0	100	1000	0
2	150	100	850	150
3	300	100	700	300
4	450	100	550	450
5	600	100	400	600
6	800	100	200	800
7	1000	100	0	1000

2.8.2 Linearity (Reportable range)

Using 2 pools one with low and the other with high concentration, five mixtures were add as shown in Table 2. These mixtures were run on ELISA in 4 replicates. The concentrations of the mixtures were obtained from the Readerfit program and this was used as the assayed concentration to plot the linearity plot using the Linchecker program. 

Table 3. Preparation of the five mixtures for linearity

Mixture	Pool	Low pool (µL)	High pool (µL)	Total Volume (µL)
1	100% low 0% high	100	0	100
2	75% low 25% high	75	25	100
3	50% low 50% high	50	50	100
4	25% low 75% high	25	75	100
5	0% low 100% high	0	100	100

2.8.3 Imprecision (Within run)

Following the protocol, extraction was performed for both low and high samples and run on ELISA in 10 replicates. The mean, standard deviation and CV% were calculated.

2.8.4 Between run imprecision

The between run imprecision was calculated by evaluating the three Biorad QC samples run throughout the project. The Levey Jennings chart was plotted to determine the acceptability of the run.

2.8.5 Sigma chart

The coefficient of variation (CV%) and bias% obtained for the low and high concentration samples were used for the sigma chart to evaluate the performance of the ELISA. The formula of the bias metrics is as below:

2.8.5 Functional Sensitivity ( Limit of Blank, limit of detection, limit of quantitation)

The Seracon (cortisol free matched serum) was run in 10 wells (10 repeats). The mean and standard deviation were calculated and used in the following equation to calculate the Limit of Blank (LoB)

LOB = mean blank + 1.645_{(SD blank).}

The low QC sample was used in 10 repeats and the following equation was used to calculate the Limit of Detection (LoD)

LOD = LOB + 1.645(SD low concentration sample).

A curve was plotted with the LOB and LOQ and the CV%, the concentration at which the CV=20% was used as the Limit of quantitation ( LOQ). 

2.9.6 Method comparison with LCMSMS

The developed ELISA was compared to the LCMS/MS by running the RCPA QAP samples and the concentrations were obtained from the Readerfit and was compared to the concentrations of the LCMSMS using Passing Bablok and Bland Altman difference plot. The Method validator program was used to plot these plots. The purpose of this was to identify constant and proportional error.

CHAPTER 3:RESULTS

3.1 Selection of extraction solvent

Hexane, MTBE and ethyl acetate were run in LCMS and ethyl acetate was selected as the best solvent selected as it showed sharp peak and had high sensitivity. The chromatograms of the three solvents are shown in Figure 1.

MTBE

Hexane

Ethyl acetates

Fig.  1  The results of LLE extractions with different solvents. Hexan then ethyl acetate and MTBE. The x axis represents the time in minutes while the y axis represents the counts

3.2 Calibration curve

Calibration curves were obtained using the standard cortisol calibrators (34- 988 nmol/L) and were fitted using a nonlinear four- parameter logistic calibration plot using the Readerfit program.. The maximum asymptotic value was 505534.04 and the minimum asymptotic value was 505292.06. The value of inflection point was 7852.3 and the slope was -0.44. The QC1 and QC3 were in the reference range but QC3 was out of the range as shown in Table  , which indicated that this experiment was not so successful. Further improvement was required.

Fig.  2  calibration curve showing the concentration of standard versus the absorbance plotted by readerfit software.

Table 4.The QC concentration from the calibration curve comparing them to the reference range for their assigned lot number.

	The mean value obtained from graph	QC information	The reference range
QC1	128.50	QC1: 40661 (127 nmol/L)	Range = 107-147
QC2	359	QC2: 40662 (427 nmol/L),	Range = 359-495
QC3	405.87	QC3: 40663 (937 nmol/L)	Range = 787-1087

The QC1 and QC3 were in the reference range but QC3 was out of the range, which indicated that this experiment was not so successful. Further improvement was required.

3.2Impression: within run

Within run imprecision was determined by calculating the CV% for the low and high concentration samples. The CV % for the low and high samples was 15.4% and 65.8 % respectively. Table 5summarises the statistical data for the two samples

Table 5:  Mean, SD and CV% for the low and high concentration samples

High values	Low values
Mean	611.5	Mean	655.9
SD	397.9	SD	100.9
CV %	65.8	CV %	15.4

3.5 Between run

The between run imprecision was assessed by analyzing the 3 QCs run across 5 weeks. This was done by plotting the Levey Jennings chart as shown in Figure 4  and analyzing  in accordance with the Westgard rules. Only few values were acceptable for all the QCs. Most of the QC values were rejected as it was outside the +/- 2SD and +/- 3SD limits. This might be due to the  random error and it can be corrected by  repeating  the assay or repeating  the controls. With the level 1 QC,  3 runs was accepted as they fell within the 2SD ( yellow line). While in level 2 QC only two run were accepted as they fell within acceptable limit and not exceeding the 2SD or 3 SD. With the level 3 QC, only two runs were accepted and the rest of run were rejected.

Levey Jennings chart

Fig. 3 Levey Jennings plot: The green line is the mean, the yellow line represents the 2SD and the red line represents 3SD.

3.3 Sensitivity Test:

LOB = mean _blank + 1.645 _{(SD blank).}

        = 117.3+(1.645*8.7389)

        =131.7

LOD = LOB + 1.645_{(SD low concentration sample).}

         = 131.7 + (1.645* 100.9)   

         = 297.7

3.7 Precision profile:

The precision profile was assessed by evaluating the LOB, LOD and LOQ and the data is shown in Table and Figure

Table 6 :Concentration of LOB, LOD, high and low values and their CV%

Fig. 4 This graph represent the precision profile. The x axis represents the concentration of the target value while the y axis represents the CV%. From this graph LOQ was determined.

In this graph the low and high values were utilized. It was clearly shown the high value was 65.8% and it  didn’t meet the imprecision and bias requirement however the low value did meet the bias and imprecision value as the CV% was 15.4%  therefore, the assay isn’t fit for purpose. LoQ value that was determined from the graph was-132.1which is very low and not reliable value for LoQ as LoQ value must be close LOD or slightly higher⁸.

Removing the high value:

Fig.5 This graph represent the precision profile. The x axis represents the concentration of the target value while the y axis represents the CV%.

In this graph the low values only were utilized and high have been removed. LoQ was determined in this graph and it was 144.2 which is higher than LoD value. However, this graph is not acceptable so it didn’t include the low and high values together to check they meet the bias and imprecision requirement to conclude that assay fit for purpose or not.

3.4 Linearity

The linearity was evaluated by plotting the assigned and assayed concentrations and was found that the ELISA was linear with the upper limit of reportable range as 511 nmol/L with a best fit polynomial of y= 312.5 +0.6002x with an intercept of 0.6 and a slope of  312.5 nmol/L. The linearity plot is shown in Figure 3.

Table 7: Assigned and assayed concentration for the linearity plot

Fig.6Linearity plot. The x axis represents the assigned concentration ( the values obtained from RCPA QAP website) and the y axis represents the assayed concentration ( the values of

3.5 Method Comparison

ELISA was compared to the LCMSMS  using Passing Bablok and difference plot. The comparison is shown in Figures7 and 8.  The correlation coefficient was 0.804  with 1.56 slope of 1.56  and an intercept of -51.  The difference plot showed  a mean difference of  225nmol/L with a 95% confidence interval of 73.1 to 376.

Fig. 7This graph represent the Passing Bablok. The x axis represents the reference methods while the y axis represents field methods.

Difference plot showed the mean bias of 225 and 73.1 to 376 of 95% confidence interval.

Fig. 8 This graph represent the difference plot. The x axis represents the mean while the y axis represents difference.(The black line depicts the bias and the dashed lines show the 95% limits of agreement)

3.6 Sigma Metrics chart

Sigma chart was used to analyse the perfromance of ELISA using the bias and CV% for the low and high concnetration samples as shown in Table . The chart as shown in Figure 7 demonstrated that both the samples were unacceptable.

Table 8 : table shows the values used to draw the sigma Metrics Chart

This table shows the values used to draw the sigma Metrics Chart

	CV %	Bias
low level	15.4	-0.66
High level	65.8	349

Fig. 9 This graph represent the sigma Metrics Chart. The x axis represents the CV % while the y axis represents the bias. Both values are not acceptable as they fall outside the acceptable range.

                                                        CHAPTER 4: DISCUSSION

Since the aim of the laboratory experiment was towards developing and validating a competitive ELISA assay for the measurement of the concentration levels of cortisol in serum and also making comparisons between the results obtained from the competitive ELISA with those obtained from the mass spectrometry TANDOM from the RCPA, it is evidently clear that by critically evaluating the two methods determination of the method which is the highly sensitive, highly reproducible, most reliable, as well as accurate and precise for quantitating cortisol concentration levels in the serum can be carried out^.. This is mainly because cortisol serum concentration levels’ measurement require to be done with specificity that is extremely high even when the concentrations are significantly low for the monitoring of the steroid hormone levels in both research and clinical laboratories to make sure there is accuracy in diagnosis for appropriate treatment and follow-up.^12. Thus, considering that the experiment needed cortisol to be extracted first it is very justifiable that the initial part of the experiment was to develop a technique that is ELISA based for cortisol extraction which started by the selection the solvents for extracting the cortisol where three of them were considered such as ethyl acetate, hexane, and MTBE and then the extracts were run on LC-MS/MS^23.. However, amongst the considered cortisol extraction solvents ethyl acetate was eventually chosen as the extraction solvent that was most preferred mainly because it exists in a non-liquid matrix as well as due to the fact that it also exists an organic solvent and cortisol which was the target extract exists as an organic soluble steroid which means that in order for cortisol’s extraction phase which should be organic to be achieved ethyl acetate would only be the most preferred solvent for extraction. Moreover, extraction of cortisol using ethyl acetate is also advantageous due to the fact that it can be easily removed completely from the extract or cortisol when there is utilization of centrifugal vacuum devices²⁴.

In order to succinctly determine the concentration of cortisol at any measurement ranging between the minimum and maximum concentration, a calibration curve was plotted using readerfit software utilizing the five parametric logistic calibration plots. Two blanks and seven standards and 3 samples of QCs were used to plot the curve. The calibration curve showed a working range of between 250nmol/Land about 750nmol/L. The results of the calibration curve implies that the ELISA method is capable of measuring cortisol within a range of the least concentration to highest concentration, that is, from the lowest concentration of 250 nmol/L to the highest concentration of 750 nmol/L.^(5-12) Therefore, the plotted calibration curve can be very essential for  the determination of the unknown concentrations of the sample solution in an experiment, since the sample solution is plotted against the observable variable on basis of their concentration and absorbance or the calibration standards of several prepared solutions. After plotting the calibration curve, unknown solution concentrations (which in this case are assumed to mean cortisol concentrations) can be determined upon placing the sample solution on the calibration curve mainly on the basis of its absorbance or on the basis of other observable variable.¹⁶ In this experiment, a calibration curve was vital for the determination of concentrations of cortisol at varied ranges of absorbance on the basis of other observable variable. ^{(7-10, 17-20)} The calibration curve is clearly showing high response values in low concentration. But, as the concentration increased the response values dropped. It is difficult to distinguish the concentration beyond 500nmol/L.

Furthermore, a consideration of the Levey Jennings chart was a clear indication that all the QCs did not effectively function its intended role due to the fact that the values for high and medium QC were undeniably within the limits which could be regarded as acceptable even though a consideration of the values for the low QC the Levey Jennings chart showed that they were undeniably not within the limits. ^{38, 39.}

This clearly indicate that there was a considerably improved techniques for developing the assay, and the results that were obtained which could not be found to be within the present limits and/or ranges could have been attributed to a number of reasons which include some errors which are inherent in the process of experimentation particularly procedures such as those involved in the extraction of the cortisol, contamination of the extraction solvent and/or the extract as well as errors in pipetting^{. (11, 25, 27)} Furthermore,the observed variations which may be considered to relatively considerable when compare to the expected limits and/or ranges may have also been as a result of the errors that may have likely occurred in course of the process through which the analyte and/or extract is labeled or transferred  as well as errors that are inherent in the calibration deviations that are integral within the equipment and/or devices used for the measurements carried out during the laboratory experiment. Thus, this implies that the standard procedures that are previously laid down should be stringently adhered to during the laboratory experiment for the purpose of making sure that the obtained results are reliable, valid and can be reproduced.³¹

During the laboratory project there was also consideration of technique validation by evaluating the accuracy, sensitivity, linearity and imprecision for the ELISA technique in order to make sure it could be appropriately and effectively used for precise measurement of cortisol concentration levels²⁸. In order to achieve this, the necessary protocols were stringently adhered to because it was regarded extremely crucial due to the fact that making conclusions and decisions concerning the validity and/or reliability of a certain procedure used to carry out scientific laboratory experiments¹³. For instance, according to Lequin⁵ clinical significant decisions could only be made regarding the analyte, whereby the degree of change that is detected is considered to the same as the total error that is allowable (TEa). ¹ This implies that bias or  accuracy indicates of disagreement between any two or multiple analytical methods and/or procedures used in the experiment which are under comparison. However, the systemic error that is detected for a particular method is recorded in terms of positive or negative bias. This implies that description of the recorded variations between the two or more methods being compared can be done using a coefficient of variation due to the fact that it usually provides a good idea with regards to the method and/or performance as well as the assay. ¹⁸ According to Lumsden¹⁸ a good method’s and/or technique’s performance is in most cases or always denoted by a CV (coefficient of variation) of below 5%; whereas whenever the CV (coefficient of variation) is equal or higher than 10%, this implies that the developed method’s and/or technique’s performance is not satisfactory. In addition, Yeh, Glock and Ryu¹³  reiterated that the upper limit of CV (coefficient of variation) for cortisol should be + 25% if the developed method’s and/or technique’s performance is good. However, the  CV (coefficient of variation)  for the results of the developed ELISA technique indicated that a CV (coefficient of variation)  of 32% was obtained with a bias of 19 % for the higher sample and CV (coefficient of variation)  of 24% and a bias of 20.7% for the lower sample. This is a clear indication that developed ELISA technique or method performance was not good or satisfactory for the analyte. Thus, in order for this analyte to be analyzed further, Sigma Chart or medical decision chart was used mainly because the achievement of the Six Sigma is undoubtedly the world class measure of the gold standard for the definition of quality. This means that performing an assay at the 3-sigma level, this is considered to be the minimum quality level acceptable; while when a method or an assay is below the 3-sigma level, adoption of a new better method needs to be implemented due to the fact that the quality of the test/analysis can’t be assured even upon repetition of the  QC runs. ¹⁷ Thus, considering figure 9 in the results chapter it can be clearly seen that  for this particular project the developed ELISA method or technique could not be accepted  for both the levels based on the Sigma chart mainly because the sigma levels were considerably low.

In addition, the results obtained from the laboratory project were also utilized in the plotting of the linearity which with regards to observed results it can be clearly seen that the linearity of the ELISA extended up to 750 nmol/L meaning that 750 nmol/L was the upper limit for detecting cortisol. Therefore, the linearity plot results are clearly indicative of the fact that there was consistency between the results obtained from the calibration curve which also indicated that the upper limit at which concentration levels of cortisol could be detected was 750nmol/L. Thus, irrespective of the fact that the developed ELISA technique or method performance was not satisfactorily good for the detection of the analyte considered in this laboratory experiment/project which is cortisol, a clear evidence of considerably high level of correlation between the two measures for the analyte such as the linearity curve as well as the calibration curve is observable.^30-32

Furthermore, the descriptions of the analyte’s lowest concentration was done in terms measures such as the sensitivity, functional/ analytical sensitivity, Limit of Blank (LoB), Limit of Detection (LoD) as well as  Limit of limit of quantitation (LoQ) and there measurement cab achieved through an assay or through the method/techniques used. For example, LoB is indicative of the fact that 95% of the measurement values that are observed upon consideration of a Gaussian distribution, whereas LoD provides a method for the estimation of bias and imprecision when the analyte’s levels are at very low concentrations which implies that it is the lowest analyte’s concentration that can be distinguished from the LoB concentration levels which implies that when compare to LoB it should always be greater than LoB.¹¹ However, when the obtained Gaussian distribution was considered, 95% of the analyte’s values were above the LoB while 5% of the analyte’s values were below LoB. Furthermore, a consideration of the LoQ means that it is lowest concentration level at which the measurement of the analyte can be done, but not detected at a point where the goals for bias and imprecision are met. Additionally, functional sensitivity was also considered which is a measure of an assay’s precision of measurement at low concentration levels of the analyte and in this project the functional sensitivity resulted in a CV (coefficient of variation) of 20%. Sometimes, the values for LoD can be equivalent to the values of LoQ but it can’t be higher. For example, if measurements the imprecision or bias of LoD are done within the TEa or total error that is allowable, LoQ can be equivalent to LoD.²¹ This means that when the results obtained in this project for  LoB, LoD and LoQ are considered considerable insights can be gained concerning the biasness and  imprecision of the method and/or analyte based on TEa or the total allowable error. Therefore, on the basis of the results obtained in this experiment, the LoQ is not equivalent to LoD, implying that the imprecision and bias doesn’t meet the total allowable error requirements. ^(9-12). The Sensitivity result was 131.7 using this equation LOB = mean blank + 1.645 (SD blank); whereas at the same the result for LOD was 297.7 using this equation LOD = LOB + 1.645(SD for a sample with low concentration of the cortisol).In figure 4showed the  low and high values were utilized. It was clearly shown the high value was 65.8% and it  didn’t meet the imprecision and bias requirement however the low value did meet the bias and imprecision value as the CV% was 15.4%  therefore, the assay isn’t fit for purpose.  LoQ value that was determined from the graph was -132.1which is very low and not reliable value for LoQ as LoQ value must be close LOD or slightly higher⁸. While in figure 5 showed the low values only were utilized and high have been removed. LoQ was determined in this graph and it was 144.2 which is higher than LoD value. However, this graph is not acceptable so it didn’t include the low and high values together to check they meet the bias and imprecision requirement to conclude that assay fit for purpose or not. The reason why the results was like this is because we used the concentration of target values instead of measured values instead of. LoQ was the lower limit of reportable range. The upper limit of this range we can get it from linearity.

The final part of the experiment was to make comparisons between the results obtained from the developed ELISA with the results which were obtained from the RCPA QAP by analyzing using the difference plot as well as the Passing Bablok. The results of Passing Bablok in figure 7 showed a regression with 1.56 slope and -51 intercept and  the correlation coefficient was 0.804. While Difference plot in figure showed the mean bias of 225 nmol/L and 73.1 to 376 of 95% confidence interval, which meant that there was relatively low level of agreement between the RCPA from QAP and the developed ELISA technique or method which showed a mean difference of 225 nmol/L. ^{(5, 22, 32)}

CHAPTER 6: CONCLUSION:

A satisfactory ELISA was developed, validated and compared to the LCMS/MS. There are many factors that should be considered when developing an assay such as the pH of the buffer, incubation time and temperature, extraction procedure, pipetting etc. Further studies should be performed to completely evaluate the performance of the ELISA by taking into account of these factors. Therefore, in clinical and research laboratories, this type of procedures are usually carried out on routine basis for the purpose of making sure comparisons between various methods or techniques used in the biomedical laboratories is done. Moreover, these procedure have also been extensively used as a protocol for the validation of a newly developed technique or method of analyte measurement in order to make sure that the obtained results are always reliable, valid and reproducible.  Thus, as new methods continue to be developed on day to day basis in the biomedical field for utilization in the clinical and research laboratories, validation of these techniques must always be prioritized despite been throughput. Finally, routine comparison of various methods and/or techniques used in the same laboratory for measurement of a similar analyte should be encouraged in order to regularly determine which of them is producing more reliable, valid and reducible results.                                             

Acknowledgement

I would like to express my gratitude to Dr. Ronda Greaves and Dr. Sonia La Vita for their supervising this experiment.

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Appendix

Coating buffer calculation :-

                   10 X PBS   10% Stock solution

                    0.1 M NaHCO₃   pH 9.6     

Using this equation:

               C₁V₁=C₂V₂

C₁= 10X                        C₂= 1 X

V₁= Required               V₂= 250

                      (1X)      X     250

V₁=———————————————– = 25mL of 10X PBS

                      (10X)

To prepare the 0.05 %  (Tween 20) from 10% (Tween 20):-

Using this equation:

                      (0.05%)      X     250mL

V₁=———————————————– =  1.25 mL of 10 % Tween 20 (V/V)

                         (10%)

Coating buffer preparation with monoclonal Antibody:

Calculation for antibody dilution:-

The procedures :-

• Prepare 2 mL of diluted antibody
• 1µL in 10 mL of coating buffer

                                                  (10X)      X    (2/10)

                                    X=———————————————– =  0.2 µL

                                                                  2

• Add 0.2 µL of coating buffer in 2 mL of coating buffer

Preparation Blocking buffer:-

The procedures :-

• Prepare 0.05 % BSA in PBS

Using this equation:

                      (0.05%)      X     4mL

V₁=———————————————– =  0.02 mL of 10% BSA

                         (10%)

So that means we will add 0.02 mL of BSA to 4 mL of 1X PBS

Preparation of Conjugate ( Cortisol-3- CMO(HRP))dilution:-

Calculation for antigen dilution:-

Dilution factor                       1 : 10,000

The procedures :-

• Prepare 1 mL of conjugate ( Cortisol-3- CMO(HRP))
• 1µL in 10 mL of PBS

               10X µL in 2 mL

                   X µL in (2/10)

                                                  (10X)      X    (2/10)

                                    X=———————————————– =  0.2 µL

                                                                  2

• Add 0.2 µL of conjugate  in 2 mL of PBS

Impression: within run

High value

	Values of absorbance	concentration
1	311993	323.9419
2	388509	98.8494
3	285373	455.4155
4	245916	731.3369
5	225318	929.0800
6	185839	1467.2288
7	311840	321.2243
8	323714	276.5652
9	261524	608.3172
10	227781	902.9952
	Mean	611.4954
	SD	397.8772
	CV %	65.8066

Low values

	Values of absorbance	concentration
1	829635	455.1
2	252287	689.7
3	248074	697.3
4	249363	694.8
5	271483	664.6
6	204408	>1001.9
7	211250	>1001.9
8	202188	>1001.9
9	192807	>1001.9
10	234568	733.8
	Mean	655.8763
	SD	100.8508
	CV %	15.3765

Calculation for LOB and LOB:

Group Name	Response Mean	Response SD	Response %CV	Calculated Mean	Calculated SD	Calculated %CV
LOD	5943060.3	1928778.172	32.4543	0.5484	0.3587	65.4134
LOB	4083183.333	1002185.268	24.5442	117.3391	8.7389	7.4476

LOQ calculation from the graph ( without removing the high value)

LoQ= (20-27.5)/0.06

        =144.2

LOQ calculation from the graph ( with removing the high value)

Values obtained from reader fit for the calibration curve ( standards and QC values):

	Response values	concentration	Concentration value
blank1	534863	0.01	<0.0100
blank1	471713	0.01	11.8
s1	341024	100	223.6
s1	447067	100	30.4
s2	350013	250	197.5
s2	317774	250	302.9
s2	327923	250	265.9
s3	316035	400	309.7
s3	292409	400	414.2
s3	277624	400	493.9
s4	285846	550	448.0
s4	267153	550	558.7
s5	231014	700	850.2
s5	235513	700	806.9
s6	234849	850	813.2
s6	245525	850	718.6
s7	207786	1000	1115.3
s7	222829	1000	935.1
QC1	422108		57.6
QC1	394238		99.4
QC2	304339		358.2
QC2	239970		766.3
QC3	294084		405.9

Calculation for Passing Bablok: