Immunology

Immunology
Name of the Patient
Professor’s Name
Results
For detecting the concentration of PSA in the serum samples of three patients, a standard curve was prepared first. The standard curve represented the optical density of known concentrations of PSA. Hence, different known concentrations of PSA were used for constructing the standard curve. Before the known or unknown concentrations of PSA were used to construct the standard curve, the optical density of a blank solution was initially noted. The optical density of the “Blank” was deducted from the optical density achieved with different standard solutions and the unknown solutions (serum samples of patients). The optical density of each standard solution was measured thrice for estimating the average absorbance (optical density) (Appendix-1). The average optical density of different standard solutions is represented in table1.
Standard Concentrations of PSA Optical Density
0 0.167
200 0.207
400 0.239
600 0.281
800 0.296
1000 0.332
1200 0.313
1400 0.342
1600 0.293
1800 0.345
2000 0.400
Table 1: Average optical density of different standard solutions of PSA
These values were used for constructing the standard curve (Fig 1). The line of best fit was constructed for extrapolating the unknown concentrations of the serum samples (PSA) as a function of optical density.

Fig 1: Standard Curve for PSA with line of best fit
Fig1 Represented the standard curve of PSA with the line of best fit. The optical densities were plotted against the Y-axis, and the PSA concentrations were plotted against the X-axis.

For each corresponding PSA concentration, there was a corresponding Optical density.
Patients Average OD x dil factor Abs – Blank   Final PSA (ng/mL)
Patient A 0.301   0.201    
Patient A (Dilute) 0.212 0.424 0.324 1619.00 16.19
Patient B 0.434   0.334    
Patient B (Dilute) 0.215 1.077 0.977 4882.33 48.82
Patient C 0.548   0.448    
Patient C (Dilute) 0.241 2.407 2.307 11532.33 115.32
Table 2: Represents the absorbance of unknown PSA concentration from serum samples of patients (with and without dilution).
The final PSA concentrations of the patients were determined by extrapolating the optical densities obtained on the Y-axis and tallying the same with the respective PSA concentrations based on the standard curve for PSA. The absorbances were noted after dilution of the PSA samples with different dilution factors.
Discussion and Conclusion
The present article reflected the assay of Prostate Specific Antigen (PSA) based on Enzyme-linked immunosorbent assay (ELISA). ELISA is an immunological technique for quantifying antigen-antibody reactions. ELISA is also used for identifying an unknown antigen. Hence, ELISA may be used for both qualitative and quantitative assays. Antigens react with antibodies either in vitro or in vivo. Antigens react with antibodies through epitope -paratope interactions. The strength of antigen-antibody reactions is dependent upon affinity and avidity of the specific antigen towards a specific antibody. ELISA is a solid-phase enzyme immunoassay for detecting an antigen in a wet sample. It is used as a popular diagnostic test for detecting various molecular markers of different diseases. In this technique, an enzyme-tagged antibody is used to assay a specific antigen. When a specific antigen binds to its respective antibody, the enzyme causes a change of color of a previously added substrate. The change of color is estimated as a function of absorbance through spectrophotometric analysis. The change of color is directly proportional to the concentration of the unknown antigen (Lequin, 2005).
ELISA is conducted by either of the two procedures. The two procedures for conducting ELISA are known as “Direct ELISA” and “Sandwich ELISA.” In the direct approach, the sample containing the unknown antigen is immobilized (by adsorption) on a polystyrene microtiter plate. The microtiter plate acts as a solid support. On the other hand, in the sandwich approach, the sample containing the unknown antigen is immobilized through capture by a specific antibody attached to the polystyrene microtiter plate. After the unknown antigen is immobilized, the enzyme-tagged antibodies (detection antibodies) are added to the microtiter plates. Bovine serum albumin is added to the microtiter plates for covering those surfaces that are not occupied by the bound antigens. The detection antibody may be directly linked to the enzyme through covalent bonds, or it might be linked with a secondary antibody (anti-antibody) through bioconjugation. On the other hand, the secondary antibody (anti-antibody) is covalently tagged to the enzyme. The microtiter plate is washed with suitable steps between each step for removing unbound or non-specifically bound antigens or antibodies. The plate is then loaded with enzymatic substrates. The conversion of these substrates into products is detected as a visible color change. The change in color is expressed as optical density (Lequin, 2005).
For detecting the concentration of an unknown antigen, a standard curve is prepared first. The standard curve represents the optical density of known concentrations of an antigen. The present paper determined the concentration of PSA in serum by ELISA analysis. Hence, different known concentrations of PSA were used for constructing the standard curve. Before the known or unknown concentrations of PSA are used to construct the standard curve, the optical density of a blank solution is initially noted. Estimating the optical density of the “blank” helps to reduce the experimental and instrumental effects that might confound the analysis (Lequin, 2005).
There are different advantages and limitations of suing ELISA as an immunological assay. First of all, ELISA is a quick and convenient approach for detecting a small amount of antigenic proteins. Moreover, the assay does not involve the use of radioisotopes. Hence, ELISA is an effective and safe method for detecting small amounts of antigen. However, the different disadvantages of ELISA include the use of monoclonal antibodies. Such antibodies are specific to a specific epitope of the antigen. However, there can be various antigenic determinants and therefore certain determinants would be underestimated. Monoclonal antibodies are often costly. On the other hand, negative controls may sometimes yield false-positive results. Such results may lead to overestimation of the concentration of the unknown antigen. False-positive results are often witnessed when the secondary antibody (anti-antibody) gets directly adsorbed on the microtiter plates. The duration of the color reaction (developed due to the interaction between the enzyme and substrate) is often short. Such issues may limit the validity and reliability of a specific assay (Kragstrup et al., 2013).
Prostate-specific antigen (PSA) is a popular marker for detecting prostate cancer. PSA is a glycoprotein enzyme and is coded by the KLK3 gene. PSA is also known as kallikrein-3 or gamma-seminoprotein. Prostate-specific antigen is a member of the kallikrein-related peptidase family and is secreted by the epithelial cells of the prostate gland. The main role of PSA is to lubricate the semen in the seminal coagulum. PSA helps the sperm to penetrate the cervical mucus during coitus. Some amount of PSA is always present in the serum of males. However, the concentration of PSA markedly increases in the event of prostate cancer. Hence, the differential diagnosis should be implemented before confirming the presence of prostate cancer in a concerned individual. However, PSA is routinely used for screening the presence of prostate cancer. Based on the estimation of PSA; patients who have prostate cancer are categorized into different risk groups (Velonas et al., 2013).
Individuals presenting with a PSA concentration of <10ng/ml is considered as a low-risk group. However, for confirming prostate cancer, they should present with a Gleason score of <6. Individuals presenting with a PSA concentration of 10ng/ml -20ng/ml is considered as the intermediate-risk group. However, for confirming prostate cancer, they should present with a Gleason score of 7. On the other hand, Individuals presenting with a PSA concentration of >20ng/ml is considered as the high-risk group. However, for confirming prostate cancer, they should present with a Gleason score of >8. PSA levels are periodically estimated after initiation of management of prostate cancer. PSA levels are estimated after every 6 to 36 months. Prostate cancer is usually treated with radiotherapy or surgical interventions. The estimation depends upon the risk stratification of prostate cancer. The success of management of prostate cancer could be estimated from the baseline PSA concentrations after initiation/completion of treatment. If there is a subsequent increase in the PSA levels above 2ng/ml, it indicates recurrence of prostate cancer (Velonas et al., 2013). The present study indicated that patients B and C were at higher risk of prostate cancer. On the other hand, patient A was at intermediate risk of prostate cancer.
However, PSA is not a confirmatory test for prostate cancer. This is because prostate specific antigen remains elevated during benign prostate hypertrophy or benign prostate hyperplasia. Moreover, different drugs like finasteride (used for treating benign prostate hyperplasia) may suppress PSA levels in the serum. Hence, the differential diagnosis must be implemented for confirming the prostate cancer. One such diagnostic test is the analysis of Free PSA/Bound PSA ratio. Most PSA in the serum is bound to plasma proteins. PSA which is not bound to plasma proteins is referred as “Free PSA,” while PSA that is bound to plasma proteins is referred as “Bound PSA.” In patients suffering from prostate cancer, the ratio of Free PSA/Bound PSA is reduced. A reduction in the ratio < 25% confirms the presence of prostate cancer in a concerned individual. Moreover, estimation of free PSA/bound PSA ratio also eliminates the necessity of frequent biopsies (Velonas et al., 2013).
References
Kragstrup, Tue W; Vorup-Jensen, Thomas; Deleuran, Bent; Hvid, Malene (2013). “A simple set of validation steps identifies and removes false results in a sandwich enzyme-linked immunosorbent assay caused by anti-animal IgG antibodies in plasma from arthritis patients.” SpringerPlus. 2 (1), 263
Lequin, R. M. (2005). “Enzyme Immunoassay (EIA)/Enzyme-Linked Immunosorbent Assay (ELISA).” Clinical Chemistry. 51 (12), 2415–8.
Velonas VM, Woo HH, dos Remedios CG, Assinder SJ (2013). “Current status of biomarkers for prostate cancer” International Journal of Molecular Sciences. 14 (6), 11034–60.

Appendix-1
  Absorbance (450nm)    
Concentration 1 2 3 Average Minus Blank
0 0.140 0.177 0.184 0.167 0.000
200 0.183 0.231 0.206 0.207 0.040
400 0.167 0.263 0.286 0.239 0.072
600 0.243 0.337 0.264 0.281 0.114
800 0.278 0.322 0.287 0.296 0.129
1000 0.304 0.331 0.360 0.332 0.165
1200 0.303 0.296 0.339 0.313 0.146
1400 0.376 0.321 0.328 0.342 0.175
1600 0.311 0.281 0.288 0.293 0.126
1800 0.368 0.359 0.308 0.345 0.178
2000 0.428 0.411 0.362 0.400 0.233
Fig: Optical densities of standard solutions of PSA with and without adjusting for blank.

Fig: Standard curve of PSA.
Appendix-2
BOV 0.078 0.087 0.107 0.091        
Blank (75μl PBS) 0.125 0.087 0.088 0.100        
        Average x dil factor Abs – Blank   Final PSA (ng/mL)
Patient A 0.321 0.287 0.294 0.301   0.201    
Patient A (Dilute) 0.246 0.209 0.181 0.212 0.424 0.324 1619.00 16.19
Patient B 0.444 0.428 0.429 0.434   0.334    
Patient B (Dilute) 0.231 0.213 0.202 0.215 1.077 0.977 4882.33 48.82
Patient C 0.585 0.502 0.557 0.548   0.448    
Patient C (Dilute) 0.248 0.226 0.248 0.241 2.407 2.307 11532.33 115.32
                 
Table: Estimation absorbance of PSA concentration of Patients serum samples