Back ground:-
Carbohydrate Antigen CA19-9 has been widely utilized for diagnosis of different kinds of cancer such as pancreatic cancer, cancer of upper gastrointestinal tract. It may also be used as an indicator of pancreatic tissue damage that may be caused by diabetes.The method of enzyme linked immunosorbent assay (ELISA) for measurement CA19-9 in sera of patients with type 2 diabetes mellitus was developed using external CA19-9 antibody (competitive sandwich ELISA technique) and found to be suitable 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.
Objectives:-
This study was performed to assess CA19-9 levels in sera of patients with type 2 diabetes mellitus with non-cancerous tissue by enzyme-linked immunosorbent assays (ELISA) and define the normal range (cutoff value) for the same marker in this patients also investigated the possible relationships of CA19-9 with fasting plasma glucose and HbA1c.This study present a new method for determination the dissociation constant (Kd) of the binding of CA19-9 to its antibody in type 2 diabetic patients using scatchard plot through development of ELISA (competitive sandwich ELISA technique).Different factors affecting this binding were extensively studied such as divalent cations, different halides, and Polyethylene (PEG-6000).
Methods:-
A case control study was conducted on eighty patients diagnosed as type 2 diabetes mellitus (T2DM) during the period from February 2015 to May 2015. Seven milliliters of blood sample was collected from each fasting subject (12 hour fast) with mean age (46.5625 ± 1.1054 years) were divided into two groups. Group Ⅰ, forty patients with type 2 diabetes mellitus have a standard error of mean duration 6.6± 0.9421 and take medication. Group Ⅱconsisted of forty patients were newly diagnosed type 2 diabetic and don't take medication, in addetion to forty healthy individuals with standard error of mean age (44.7± 1.4539) were classified as control group.Glycate hemoglobin was measured in whole blood by NycoCard reader instrument, while FBS, lipid profile, Urea serum, serum ccreatinine, C-reactive protein, CA19-9 level, determination of Antigen Affinity, protein concentration using Bradford method were measured in serum by Chemistry Analyzer, NycoCard reader instrument ELISA technique and Spectrophotometer.
Results:-
The diabetic groups (with treatment & newly diagnosed) showed a significant elevation of serum CA19-9 protein in comparison with control group (p< 0.001), while there is no significant difference observed between the two diabetic groups (p=0.308). Serum CA19-9 concentration of 49.5 U/ml was used as cut off value for the diagnosis of type 2 diabetes mellitus, the sensitivity is 88.8% and specificity is 77.5%The converter factor 1.010388 can be used to convert the concentration of carbohydrate antigen CA19-9 from (U/ml) to (µg/ml).The dissociation constant (Kd) of CA19-9 for standard and patient was 15.6006 U/ml and 12.5313 U/ml respectively, while for healthy individual is 4.1271 U/ml.The current study proves there is an effect for divalent cation which showed that the binding process is sensitive for the cations existence, it is found that Mn (II) at concentration 0.15M increases the binding more than the other divalent cations, whereas Cu (Ⅱ) in the same concentration increased the binding less than other divalent cations.The effect of different halides (NaI, NaCl and NaF) on CA19-9 Antigen-Antibody binding in incubation medium that contain sodium halides which causes activation in the percent of binding as the following sequence:
NaF > NaCl > NaI
Study the effect of divalent cations and different halides on the binding of carbohydrate antigen CA19-9 to its antibody is useful in the future work to obtain the maximum bind and create an optimum conditions for this binding.Polyethylene (PEG-6000) affect negatively on CA19-9 Antigen-Antibody binding.
Conclusion:-
The CA19-9 level not only confined to identify the cancerous tissue but it rise in non-cancerous cases also such as type 2 diabetes mellitus as a result for pancreatic tissue damage that may be caused by diabetes. And the dissociation constant of the interaction between antibody and antigen for the same tumor marker can be calculated using a new method from the data of competitive sandwich enzyme linked immunosorbent assay (ELISA) by Scatchard plot.
