ISSN 2320-5407 International Journal of Advanced Research (2016), Volume 4, Issue 4, 710-719

Russul R. Al-Hamaoy. Background:-In order to quantify the interactions between molecules of biological interest, the determination of dissociation constants (Kd) is essential. Several methods are known for calculating these constants, most of which are based on linearization procedures, such as the Scatchard (1949) plot, Lineweaver and Burk (1934) plot, etc. This linearization does not require direct ligand labelling or the absolute concentration of the complex and is, therefore, especially suitable for Kd determination from an enzyme-linked immunosorbent assay (ELISA). Objectives: Present a new method for the determination of a dissociation constant (Kd) of the binding of CA19-9 to its antibody in type 2 diabetic patients using Scatchard plot through development of ELISA. Methods:This study included eighty individuals with mean age (46.5 ± 1.14 years) were divided into two groups. Group I,forty patients with type II diabetes mellitus have a mean duration 6.6± 0.94.Group IIconsisted of forty healthy individuals were classified as control group. CA19-9 level were measured in serum by ELISA technique.Select the highest value from the first group and other value within the normal range chosen from the second group in addition to the CA19-9 standard (100U/ml) to determine the dissociation constant (kd) using external CA19-9 monoclonal antibody and calculated by Scatchard plot. Results:The dissociation constant of the interaction between antibody and antigen from the data of direct, non-competitive enzyme linked immunosorbent assays (ELISA) by Scatchard plot is (15.6006, 12.5313 and 4.1271 U/ml) for Standard (100 U/ml), Patient (99.568 U/ml) and Control (23.494 U/ml) respectively. Conclusion:-A simple linearization procedure developed to determine the dissociation constant of the interaction between antibody and antigen from the data of direct, non-competitive enzyme linked immunosorbent assays (ELISA) by Scatchard plot.


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cancer [2] . Furthermore, this antigen has a high value during the diagnosis of pancreatic cancer, because it has a sensitivity of 70-90% and a specificity of 68-91% [3] .
Diabetes has been claimed to be a risk factor for pancreatic cancer, which is increasing its incidence and has one of the lowest survival rates of all cancers [10] .
In order to quantify the interactions between molecules of biological interest, the determination of dissociation constants (Kd) is essential. Several methods are known for calculating these constants, most of which are based on linearization procedures, such as the Scatchard (1949) plot [11] , Lineweaver and Burk (1934) plot [12] , etc. The main advantage of linearization methods is their simplicity. Previously, a simple linearization procedure was developed to determine the Kd of antigen -antibody (Ag -Ab) interactions [13,14] . This linearization does not require direct ligand labelling or the absolute concentration of the complex and is, therefore, especially suitable for Kd determination from an enzyme-linked immunosorbent assay (ELISA). Lee et al., 1996 obtained the dissociation constant (Kd) of antigen-antibody complex using Scatchard equation through direct ELISA [15] .

Sang-Han
In this study we present a new method for the determination of a dissociation constant (Kd) of the binding of CA19-9 to its antibody in type 2 diabetic patients using Scatchard plot through development of directnon-competitive enzyme linked immunosorbent assays (ELISA).

Methods:-
The study was executed during the term from February 2015 to May 2015. The study included eighty individuals (35 male and 45 female) have a standard error of mean age (mean ± SE) 46.5 ± 1.14 years, were divided into two groups. Group Ⅰ,forty patients with type Ⅱ diabetes mellitus have a mean duration 6.6± 0.94. Group Ⅱconsisted of forty healthy individuals were classified as control group.
Blood sample was collected from all 80 subjects from Al-Imamain Al-Kadhimain Medical City, Baghdad, Iraq. The approval of the Al-Nahrain University/ college of Medicine Research Ethics Committee.
Patients with any malignancies or suffered from pancreatic, thyroid, liver, and renal diseases in their medical history, pregnant women and smokers were excluded. Blood samples were centrifuged at 3000 rpm and serum was stored at -20 o C. Serum CA 19-9 level was measured by monoclonal antibody Enzyme Linked Immuno Sorbent Assay (ELISA) technique. Select the highest value from the first group and other value within the normal range chosen from the second group in addition to the CA19-9 standard (100U/ml) to determine the dissociation constant (kd) using external CA19-9 monoclonal antibody and calculated by Scatchard plot. The data obtained was analyzed by Microsoft excel 2013.

Results:-
Standard solution (0, 10, 50, 100, 250, 500U/ml) of CA 19-9 ELISA kit used to determine the standard curve as figure (1): The original concentration of Monoclonal antibody solution was 100 µg /100 µl, as a result when made up the volume with 0.01M Phosphate Buffer Saline to 1 ml the concentration become 10 µg/ml = 0.01 mg/ml and used the last as a stock solution. To find the concentration of Ab in each well used the following equation:- B= The bound of CA19-9 to its immobilized antibody (B) that can be obtaining from ELISA curve. F= the free concentration of CA19-9 which represent the first incubation in solution that can be deduced from the total binding (TB) of CA19-9 obtained from the standard curve of sandwich ELISA and B. Where The same previous equations were applied for patient has a CA19-9 level (99.568U/ml) and for healthy individual has a CA19-9 level (23U/ml), which were gave the following results:-

Discussion:-
Enzyme-linked immunosorbent assay (ELISA) is one of immunoassay methods using antibody to capture an antigen (target antigen) then using an enzyme labeled antibody for estimate the antigen amount. (16) It is widely used as a clinical diagnostic tool to detect a vast range of diseases from infection diseases to cancer biomarkers. ELISA instrument is described as a versatile, precise, quantifiable and sensitive diagnostic method. (17,18) Several methods are known for calculating dissociation constant, most of which are based on linearization procedures, such as the Scatchardplot (1949) (11) , Lineweaver and Burk (1934) plot (12) , etc.
The present study suggest a simple linearization procedure developed to determine the obvious dissociation constant of the interaction between antibody and antigen from the data of direct, non-competitive enzyme linked immunosorbent assays (ELISA) by Scatchard plot. FerencOrosz and JuditOva´di concluded no linearization procedure has been described for determination of dissociation constant (Kd) from displacement ELISA (14) .
Liliom, K et al. described a linearization procedure for determination of dissociation constants (kd) of antigenantibody interaction using data from the enzyme-linked immunosorbent assays (ELISA). (13) Katsumi discussed the impact of dissociation constant on the standard curve from the below equation of antigenantibody interaction, And conclude that the standard curve shift to right depending on the value of dissociation constant (Kd), which indicates the sensitivity of assay is related to Kd. Therefore if Kd is small (the affinity constant is large), the sensitivity becomes excellent. (16) Funding:- The research was funded by College of Medicine, Al-Nahrain University.