Claims:WE CLAIM:

1. A simple diagnostic method for the early detection of gestational diabetes during the first trimester or at the time of confirmation of pregnancy, the claimed method comprises of:

a. collecting the serum sample of a subject;
b. detecting ZnT8-PEP specific autoantibody isotope IgA by indirect ELISA using polyepitopepeptide of sequence “NH2-NKDQCPRERPEELEGGGGTAASRDSGGGGESPVDQDPD-COOH and measuring absorbance to get ZnT8 IgA (O.D450);
c. detecting ZnT8-PEP specific autoantibody isotope IgG by indirect ELISA using polyepitopepeptide of sequence “NH2-NKDQCPRERPEELEGGGGTAASRDSGGGGESPVDQDPD-COOH and measuring absorbance to get ZnT8 IgG (O.D450);
d. detecting total immunoglobulin isotope IgA by sandwich ELISA using anti-human IgA antibodies and measuring absorbance to get Total IgA (O.D450);
e. detecting total immunoglobulin isotope IgG by sandwich ELISA using anti-human IgG antibodies and measuring absorbance to get Total IgG (O.D450);
f. calculating Arbitrary Units (AU) = ZnT8 IgA (O.D450)/ Total IgA (O.D450)
ZnT8 IgG (O.D450) /Total IgG (O.D450)
g. comparing AU with cutoff value of 1.6AU wherein if the AU is greater than cutoff value indicates no risk for developing GDM and if the AU is lesser than cutoff value indicates high risk for developing GDM.

2. The method as claimed in claim 1 wherein the said cut-off value is derived from positive control for Total IgA, Total IgG, ZnT8 IgA and ZnT8 IgG.

3. The method as claimed in claim1 wherein the detection of ZnT8-PEP specific autoantibody isotope IgA by indirect ELISA using the Polyepitopepeptide of sequence “NH2-NKDQCPRERPEELEGGGGTAASRDSGGGGESPVDQDPD-COOH” comprises of following sequential steps.

a. coating wells in ELISA plate with the polyepitopepeptide of sequence “NH2-NKDQCPRERPEELEGGGGTAASRDSGGGGESPVDQDPD-COOH, followed by incubation overnight at 40C and the next day washing thrice using wash buffer(1xPBS with 0.05% Tween-20, 150µl in each well) and pat dry
b. blocking wells with blocking buffer (1% BSA in 1X PBS, 25µl per well) for 3 hour at room temperature followed by washing the wells and pat dry
c. adding diluted serum samples (1:1000 dilution) into the blocked wells followed by incubation overnight at 370C and washing the wells thrice using wash buffer (1xPBS with 0.05% Tween-20, 150µl in each well followed by pat dry.
d. adding HRP-conjugated anti-human IgA (1:60,000 dilution) in to the wells followed by incubation for 3hours at 4˚C and washing the wells thrice using wash buffer (1xPBS with 0.05% Tween-20, 150µl in each well, 150µl in each well) followed by pat dry.
e. adding TMB (Tetramethylbenzidine)/H2O2 (Hydrogen peroxide) in to the wells followed by incubation for 30 minutes at room temperature in dark
f. adding 2N sulphuric acid in to the wells for arresting the reaction and measuring OD at 450nm using ELISA plate reader to obtain ZnT8 IgA (O.D450).

4. The method as claimed in claim1 wherein the detection of ZnT8-PEP specific autoantibody isotope IgG by indirect ELISA using the Polyepitopepeptide of sequence “NH2-NKDQCPRERPEELEGGGGTAASRDSGGGGESPVDQDPD-COOH” comprises of following sequential steps.

a. coating wells in ELISA plate with the Polyepitopepeptide of sequence “NH2-NKDQCPRERPEELEGGGGTAASRDSGGGGESPVDQDPD-COOH, followed by incubation overnight at 40C and the next day washing thrice using wash buffer(1xPBS with 0.05% Tween-20, 150µl in each well) and pat dry
b. blocking wells with blocking buffer (1% BSA in 1X PBS, 25µl per well) for 3 hour at room temperature followed by washing the wells and pat dry
c. adding serum samples (1:1000 dilution) into the blocked wells followed by incubation overnight at 370C and washing the wells thrice using wash buffer(1xPBS with 0.05% Tween-20, 150µl in each well) followed by pat dry.
d. adding HRP-conjugated anti-human IgG (1:60,000dilution) in to the wells followed by incubation for 3hours at 4˚C and washing the wells thrice using wash buffer(1xPBS with 0.05% Tween-20, 150µl in each well) followed by pat dry.
e. adding TMB (Tetramethylbenzidine)/H2O2(Hydrogen peroxide) in to the wells followed by incubation for 30 minutes at room temperature in dark
f. adding 2N sulphuric acid in to the wells for arresting the reaction and measuring OD at 450nm using ELISA plate reader to obtain ZnT8 IgG (O.D450).

5. The method as claimed in claim1 wherein the detection of total immunoglobulin isotope IgA by sandwich ELISA using anti-human IgA antibodies comprises of following sequential steps.

a. coating wells in ELISA plate with the anti-human IgA, followed by incubation overnight at 40C and the next day washing thrice using wash buffer (1xPBS with 0.05% Tween-20, 150µl in each well) and pat dry
b. blocking wells with blocking buffer (1% BSA in 1X PBS, 25µl per well) for 3 hour at room temperature followed by washing the wells and pat dry
c. adding serum samples (1:1000 dilution) into the blocked wells followed by incubation overnight at 40C and washing the wells thrice using wash buffer(1xPBS with 0.05% Tween-20, 150µl in each well) followed by pat dry.
d. adding HRP-conjugated anti-human IgA in to the wells followed by incubation for 3hoursat 4˚C and washing the wells thrice using wash buffer(1xPBS with 0.05% Tween-20, 150µl in each well) followed by pat dry.
e. adding TMB (Tetramethylbenzidine)/H2O2(Hydrogen peroxide) in to the wells followed by incubation for 30 minutesat room temperature in dark
f. adding 2N sulphuric acid in to the wells for arresting the reaction and measuring OD at 450nm using ELISA plate reader to obtain Total IgA (O.D450).

6. The method as claimed in claim1 wherein the detection of total immunoglobulin isotope IgG by sandwich ELISA using anti-human IgG antibodies comprises of following sequential steps.

a. coating wells in ELISA plate with the anti-human IgG, followed by incubation overnight at 40C and the next day washing thrice using wash buffer(1xPBS with 0.05% Tween-20, 150µl in each well) and pat dry
b. blocking wells with blocking buffer (1% BSA in 1X PBS, 25µl per well) for 3 hour at room temperature followed by washing the wells and pat dry
c. adding serum samples (1:1000 dilution) into the blocked wells followed by incubation overnight at 40C and washing the wells thrice using wash buffer(1xPBS with 0.05% Tween-20, 150µl in each well) followed by pat dry.
d. adding HRP-conjugated anti-human IgG in to the wells followed by incubation for 3hours at 4˚C and washing the wells thrice using wash buffer(1xPBS with 0.05% Tween-20, 150µl in each well) followed by pat dry.
e. adding TMB (Tetramethylbenzidine)/H2O2(Hydrogen peroxide) in to the wells followed by incubation for 30 minutes at room temperature in dark
f. adding 2N sulphuric acid in to the wells for arresting the reaction and measuring OD at 450nm using ELISA plate reader to obtain Total IgG (O.D450).

Dated this 11th day of JAN 2022

For UNIVERSITY OF MADRAS
By its Patent Agent

Dr.B.Deepa
IN/PA 1477
, Description:Form 2

THE PATENT ACT, 1970
(39 of 1970)
&
THE PATENT RULES, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)

“A SIMPLE ZnT8-PET BASED DIAGNOSTIC TEST TO DETECT EARLY GESTATIONAL DIABETES”

in the name of UNIVERSITY OF MADRAS an Indian nationals having address at UNIVERSITY OF MADRAS, CHEPAUK, CHENNAI-600005, TAMIL NADU, INDIA

The following specification particularly describes the invention and the manner in which it is to be performed

FIELD OF THE INVENTION:
The field of the invention is the diagnosis of gestational diabetes. More particularly the present invention relates to a simple diagnostic method for the early detection of gestational diabetes during the first trimester or at the time of confirmation of pregnancy.
BACKGROUND OF THE INVENTION:
The prevalence of gestational diabetes mellitus (GDM), one of the most common metabolic disorders during pregnancy, is increasing worldwide. Till date oral glucose tolerance test (OGTT) and glycated hemoglobin (HbA1c) are routinely used for the diagnosis of GDM. Pregnant women can develop GDM at any trimester. While OGTT and HbA1c can be used to diagnose GDM, till date, to the best of our knowledge, we do not have any test to predict late trimester GDM in the first trimester (when the pregnant women first visits the clinic to confirm her pregnancy and is still normoglycemic).

The existing state of art to diagnosis gestational diabetics is as follows.
1. Oral Glucose Tolerant Test (OGTT)- Estimation of fasting and 2hrs blood glucose levels post consumption of 75g glucose at 3rd trimester during pregnancy.
2. Glycated Haemoglobin (HbA1c)- Determination of the percentage of glycated haemoglobinin the circulation once every three months.
3. Glucometer: Estimationof fasting, Post-Prandial and Random Blood glucose levels at any point during pregnancy.

Fasting Blood Glucose (FBG) and HbA1c levels are often tested during first trimester or at the time of confirmation of pregnancy in order to diagnose existing diabetes. However, OGTT is performed only during the 3rd trimester, leaving a large proportion of pregnant women undiagnosed for GDM until then. These methods have limited application in predicting late-stage GDM. According to the American Diabetes Association [ADA] guidelines for GDM FBG levels of <95mg/dl, OGTT-2hr levels of < 120mg/dl and HbA1c percentage of <6% are considered as normal glycemic levels in GDM. As observed in our study, even though, FBG, HbA1c and fasting OGTT levels were increased in GDM patients when compared to control group, these levels were within normal range. Hence, these parameters do not show utility in predicting late-stage GDM. Thus, there exists a need in the state of art to develop a novel diagnostic method for the early detection of gestational diabetes during the first trimester or at the time of confirmation of pregnancy.

OBJECT OF THE INVENTION:

The main object of the present invention is to develop a novel and simple diagnostic method for the early detection of gestational diabetes during the first trimester or at the time of confirmation of pregnancy.

Another object of the present invention is to develop a simple diagnostic method for the early detection of gestational diabetes comprising of detecting ZnT8-PEP specific autoantibody isotope IgA, ZnT8-PEP specific autoantibody isotope IgG, total immunoglobulin isotope IgA&total immunoglobulin isotope IgG in a sample and arriving at an arbitrary value based on characterized calculation and comparing with cutoff value to predict the high risk or no risk for developing GDM.

Yet another object of the present invention is to formulate a simple blood test to detect pre-GDM women soon after their conception.

Further object of the present invention is to utilize the developed diagnostic test for early detection of gestational diabetes during the first trimester or at the time of confirmation of pregnancy.

BRIEF DESCRIPTION OF DRAWINGS:

Figure 1 illustrates the work flow of the present invention.

SUMMARY OF THE INVENTION:

The present invention discloses a simple diagnostic method for the early detection of gestational diabetes during the first trimester or at the time of confirmation of pregnancy. The method of the present invention comprises of following steps.
a. collecting the serum sample of a subject;
b. detecting ZnT8-PEP specific autoantibody isotope IgA by indirect ELISA using polyepitopepeptide of sequence “NH2-NKDQCPRERPEELEGGGGTAASRDSGGGGESPVDQDPD-COOH and measuring absorbance to get ZnT8 IgA (O.D450);
c. detecting ZnT8-PEP specific autoantibody isotope IgG by indirect ELISA using polyepitopepeptide of sequence “NH2-NKDQCPRERPEELEGGGGTAASRDSGGGGESPVDQDPD-COOH and measuring absorbance to get ZnT8 IgG (O.D450);
d. detecting total immunoglobulin isotope IgA by sandwich ELISA using anti-human IgA antibodies and measuring absorbance to get Total IgA (O.D450);
e. detecting total immunoglobulin isotope IgG by sandwich ELISA using anti-human IgG antibodies and measuring absorbance to get Total IgG (O.D450);
f. calculating Arbitrary Units (AU) = ZnT8 IgA (O.D450)/ Total IgA (O.D450)
ZnT8 IgG (O.D450) /Total IgG (O.D450)
g. comparing AU with cutoff value of 1.6AU in which if the AU is greater than cutoff value indicates no risk for developing GDM and if the AU is lesser than cutoff value indicates high risk for developing GDM.
Detailed description of the invention:
The present invention discloses a simple diagnostic method for the early detection of gestational diabetes during the first trimester or at the time of confirmation of pregnancy.
Zinc Transporter 8 (ZnT8) protein is a member of the zinc transporter family and is coded by the solute carrier family 30 member 8 gene (SLC30A8). ZnT8 is predominantly expressed in pancreatic beta-cells and is involved in zinc transport. Zinc in turn is involved in insulin storage, assembly and secretion. ZnT8 was first discovered as an autoantigen associated with Type-1 Diabetes (T1DM). Interestingly, an earlier genome-wide association study showed an association between a single nucleotide polymorphism in SLC30A8 withT2DM.In our lab, we conducted a bioinformatic study to identify putative B-cell epitopes in this autoantigen. The epitopes were identified using four different softwares. Out of 33 epitopes identified, only 3 epitopes were predicted by all the four softwares. A synthetic polypeptide, coding for these three epitopes connected by tetra-glycine linkers was synthesized, and was called as ZnT8-PEP (Polyepitopepolypeptide).

The sequence of the ZnT8-PEP is, “NH2-NKDQCPRERPEELEGGGGTAASRDSGGGGESPVDQDPD-COOH”.
Circulating AAbs against ZnT8-PEP was measured in both TIDM and T2DM subjects by ELISA. Our results indicated significantly reduced levels of circulating AAbs against ZnT8-PEP in both T1DM and T2DM patients, compared to control subjects. Since, GDM is characterized by combined insulin resistance and deficiency, we hypothesized that AAbs against ZnT8-PEP would also be altered in GDM. Thus, as a next step, we estimated circulating AAbs against ZnT8-PEP in two groups of pregnant women: 1. Women who remainednormoglycemic throughout their pregnancy (Control group) and 2. Women who were normoglycemic at the time of recruitment (1st trimester) but later developed GDM in the 2nd /3rdtrimester (Late GDM/GDM-L). As per our hypothesis, we found increased levels of IgG and decreased levels of IgA in the pre-GDM group compared to controls (after adjusting for total IgG and IgA levels). Next, the IgA/IgG ratio was calculated and was found to be significantly lower in the pre-GDM group, compared to control.By performing AUC analysis, we derived a cut-off value of 1.6 by which we were able to predict pre-GDM with 80% specificity and 70% sensitivity.

The detection of circulating Anti- ZnT8- Immunoglobin G (IgG) and Immunoglobin A (IgA) is a novel approach in the diagnosis and managementofpatientswith GDM.We plan todevelopthesefindingsasan over-the-counter diagnostic kit which would be available to lay persons were by the specimen can be directly collected, test performed and interpreted by user. The availability of this kit would be of great potential for patients to seek earlier diagnosis and subsequent management of GDM.

The novelty of this procedure can be drawn on two levels. One, the peptide that was used in this invention was first identified in our laboratory using several bioinformatic tools. Second, the idea of predicting late-stage GDM in the 1st trimester is conceptually new. To the best of our knowledge currently no test is available in the market to diagnose high risk pre-GDM subjects in their first trimester of pregnancy.

The cut-off value which was used to determine high risk-GDM women was derived using two independent ELISA experiments which were unique in their own way. Firstly, the ZnT8-specific isotypes were determined in the control and GDM-L groups. Secondly, ZnT8-specific isotype values were normalized by calculating the total isotype levels in both groups. Since, one isotype was significantly increased and another decreased, these values were incorporated into a single ratio which was then used to derive the cut-off value. Because of the incorporation of an additional derivative this assay becomes a unique assay.

With the increasing incidence of GDM worldwide, it's prediction at an early-stage of pregnancy is of great importance. GDM is preventable if we can identify and treat high risk individuals. GDM is currently diagnosed through biochemical tests at the antenatal screening. Only a few potential biomarkers for the prediction of GDM have been described till date, but none have been translated to clinical diagnosis. We have discovered a peptide which can be used for prognosis of GDM in 1sttrimester women by a simple blood test. Serum ZnT8-specific IgG and IgA were first estimated in the control and GDM women. Even though, ZnT8 is considered to be an autoantigen, to our surprise, IgG and IgA autoantibodies against ZNT8-PEP were significantly increased and decreasedrespectively, in late-stage GDM compared to the control group. These observations indicated ZnT8-PEP specific IgG and IgA could be used as a potential prognostic factor for late-stage GDM identification. In order to increase the specificity and sensitivity of the assay, the ratio between ZnT8-PEP specific IgG and -IgA and total IgG and IgA were used in predicting pre-GDM. These findings would be used to develop an over-the-counter sero-diagnostic test, which can be performed and interpreted by the user.

GDM is one of the most common metabolic disorders in pregnancy worldwide. Uncontrolled GDM may lead to several complications both in the mother and the offspring including postpartum haemorrhage and foetal macrosomia. Therefore, identification of pre-GDM during the first trimester or at the time of confirmation of pregnancy is essential. The existing diagnostic methods are only used to detect hyperglycemia in real time but are limited in its application as prognostic methods. To this end, the above procedure is proposed as a diagnostic method to identify highrisk GDM subjects.

Study Design: Longitudinal observational study
Study groups:
1. Control group- Normoglycemic pregnant women (Control; n=100)
2. Pre-GDM group - Women who were normoglycemic in the 1st trimester but later developed GDM in their 2nd/3rd trimester (GDM-L; n=100).
Inclusion criteria and Exclusion criteria:
The inclusion criteria were healthy pregnant women with no GDM at the time of recruitment (between 18 to 39 years of age). The exclusion criteria were patients who had T2DM or GDM (or history of GDM in previous pregnancy).
Ethical approval:
The study was approved by the institutional ethical committee at ESIC Medical College, KK Nagar, Chennai (Ref No-08/27/10/2014). Written informed consent was obtained from all the study participants. This work was carried out in accordance with the code of ethics of the world medical association (Declaration of Helsinki).
Procedure:
Estimation of ZnT8-specific autoantibodies
1. 1µg/ml of peptide was prepared using 1X PBS, and 25µl was added per well in a 96- well ELISA (Corning® 96 Well Half-Area Microplate, USA) plate and incubated at 4˚C, overnight.
2. The next day, the plate was washed thrice using wash buffer (1xPBS with 0.05% Tween-20, 150µl in each well) and patted dry.
3. The plate was blocked by adding blocking buffer (1% BSA in 1X PBS, 25µl per well) and was incubated for 3 hrs at room temperature, followed by washing (as mentioned in step 2).
4. 25µl serum samples (1:1000 dilution) was added to each well and was incubated at 4˚C, overnight.
5. The plate was then washed using wash buffer thrice and patted dry (as mentioned in step 2).
6. 25µl HRP-conjugated anti-human IgA and IgG (1:60,000dilution) (IgG-Cat. No- A0420, Sigma, USA and IgA-Cat. No. GX-6601PC1H, Puregene) was added as secondary antibodies and incubated for 3 hrs at 4˚C, followed by washing (as mentioned in step 2).
7. 25µl of 1X TMB (Tetramethylbenzidine)/H2O2(Hydrogen peroxide) substrate was then added to each well and incubated for 30 minutes at room temperature in dark.
8. The reaction was stopped by adding 25µl of 2N H2SO4 to each well and absorbance at 450nm was measured. The final antibody titre was reported as OD450.
9. In parallel experiment, for each sample, 25µl of anti-human IgG (Catno:2040-01, Soutern Biotech, 1:1,60,000) or IgA (Clone, Catno:2050-01, Southern Biotech, 1:1,60,000) was coated and incubated overnight. After washing, the plates were blocked using blocking buffer (1% BSA in 1X PBS, 25µl per well) for 3 hrs at room temperature.
10. The plate was then washed using wash buffer thrice and patted dry (as mentioned in step 2).
11. 25µl of diluted serum (1:1000 dilution) was added and incubated overnight at 4˚C.
12. The plate was then washed using wash buffer thrice and patted dry (as mentioned in step 2).
13. 25µl of HRP conjugated anti-human IgG (Catno: A6029-1ML, Sigma Alrich, 1:10,000) or IgA (Clone, Cat No: GX-6601PC1H, Puregene, 1:20,000) was added and incubated for 3 hrs at 4˚C, followed by washing (as mentioned in step 2). 25µl of 1X TMB (Tetramethylbenzidine)/H2O2(Hydrogen peroxide) substrate was then added to each well and incubated for 30 minutes at room temperature in dark.
14. After the incubation period the reaction was stopped and readings were taken as mentioned in step8.
Arbitrary Units (AU) = ZnT8 IgA (O.D450)/ Total IgA (O.D450)
ZnT8 IgG (O.D450) /Total IgG (O.D450)
15. The Area Under the Curve (AUC) was determined using SPSS software where, an area greater than 0.7 was obtained with 80% specificity and 70% sensitivity derived at a cut-off of 1.6 AU.

Figure 1 illustrates the work flow of the present invention.
In one of the preferred embodiment, the present invention shall disclose a simple diagnostic method for the early detection of gestational diabetes during the first trimester or at the time of confirmation of pregnancy. The method of the present invention comprises of following steps.
a. collecting the serum sample of a subject;
b. detecting ZnT8-PEP specific autoantibody isotope IgA by indirect ELISA using polyepitopepeptide of sequence “NH2-NKDQCPRERPEELEGGGGTAASRDSGGGGESPVDQDPD-COOH and measuring absorbance to get ZnT8 IgA (O.D450);
c. detecting ZnT8-PEP specific autoantibody isotope IgG by indirect ELISA using polyepitopepeptide of sequence “NH2-NKDQCPRERPEELEGGGGTAASRDSGGGGESPVDQDPD-COOH and measuring absorbance to get ZnT8 IgG (O.D450);
d. detecting total immunoglobulin isotope IgA by sandwich ELISA using anti-human IgA antibodies and measuring absorbance to get Total IgA (O.D450);
e. detecting total immunoglobulin isotope IgG by sandwich ELISA using anti-human IgG antibodies and measuring absorbance to get Total IgG (O.D450);
f. calculating Arbitrary Units (AU) = ZnT8 IgA (O.D450)/ Total IgA (O.D450)
ZnT8 IgG (O.D450) /Total IgG (O.D450)
g. comparing AU with cutoff value of 1.6AU in which if the AU is greater than cutoff value indicates no risk for developing GDM and if the AU is lesser than cutoff value indicates high risk for developing GDM.

As per the invention, in the method, the cut-off value is derived from positive control for Total IgA, Total IgG, ZnT8 IgA and ZnT8 IgG.
In accordance with the invention, in the method, the detection of ZnT8-PEP specific autoantibody isotope IgA is determined by indirect ELISA using the Polyepitopepeptide of sequence “NH2-NKDQCPRERPEELEGGGGTAASRDSGGGGESPVDQDPD-COOH” comprises of following sequential steps.
a. coating wells in ELISA plate with the polyepitopepeptide of sequence “NH2-NKDQCPRERPEELEGGGGTAASRDSGGGGESPVDQDPD-COOH, followed by incubation overnight at 40C and the next day washing thrice using wash buffer(1xPBS with 0.05% Tween-20, 150µl in each well) and pat dry
b. blocking wells with blocking buffer (1% BSA in 1X PBS, 25µl per well) for 3 hour at room temperature followed by washing the wells and pat dry
c. adding diluted serum samples (1:1000 dilution) into the blocked wells followed by incubation overnight at 370C and washing the wells thrice using wash buffer (1xPBS with 0.05% Tween-20, 150µl in each well followed by pat dry.
d. adding HRP-conjugated anti-human IgA (1:60,000 dilution) in to the wells followed by incubation for 3hours at 4˚C and washing the wells thrice using wash buffer (1xPBS with 0.05% Tween-20, 150µl in each well, 150µl in each well) followed by pat dry.
e. adding TMB (Tetramethylbenzidine)/H2O2 (Hydrogen peroxide) in to the wells followed by incubation for 30 minutes at room temperature in dark
g. adding 2N sulphuric acid in to the wells for arresting the reaction and measuring OD at 450nm using ELISA plate reader to obtain ZnT8 IgA (O.D450).

According to the invention, in the method, the detection of ZnT8-PEP specific autoantibody isotope IgG is determined by indirect ELISA using the Polyepitopepeptide of sequence “NH2-NKDQCPRERPEELEGGGGTAASRDSGGGGESPVDQDPD-COOH” comprises of following sequential steps.
a. coating wells in ELISA plate with the Polyepitopepeptide of sequence “NH2-NKDQCPRERPEELEGGGGTAASRDSGGGGESPVDQDPD-COOH, followed by incubation overnight at 40C and the next day washing thrice using wash buffer(1xPBS with 0.05% Tween-20, 150µl in each well) and pat dry
b. blocking wells with blocking buffer (1% BSA in 1X PBS, 25µl per well) for 3 hour at room temperature followed by washing the wells and pat dry
c. adding serum samples (1:1000 dilution) into the blocked wells followed by incubation overnight at 370C and washing the wells thrice using wash buffer(1xPBS with 0.05% Tween-20, 150µl in each well) followed by pat dry.
d. adding HRP-conjugated anti-human IgG (1:60,000dilution) in to the wells followed by incubation for 3hours at 4˚C and washing the wells thrice using wash buffer(1xPBS with 0.05% Tween-20, 150µl in each well) followed by pat dry.
e. adding TMB (Tetramethylbenzidine)/H2O2(Hydrogen peroxide) in to the wells followed by incubation for 30 minutes at room temperature in dark
h. adding 2N sulphuric acid in to the wells for arresting the reaction and measuring OD at 450nm using ELISA plate reader to obtain ZnT8 IgG (O.D450).

As per the invention, in the method, the detection of total immunoglobulin isotope IgA is determined by sandwich ELISA using anti-human IgA antibodies comprises of following sequential steps.

a. coating wells in ELISA plate with the anti-human IgA, followed by incubation overnight at 40C and the next day washing thrice using wash buffer (1xPBS with 0.05% Tween-20, 150µl in each well) and pat dry
b. blocking wells with blocking buffer (1% BSA in 1X PBS, 25µl per well) for 3 hour at room temperature followed by washing the wells and pat dry
c. adding serum samples (1:1000 dilution) into the blocked wells followed by incubation overnight at 40C and washing the wells thrice using wash buffer(1xPBS with 0.05% Tween-20, 150µl in each well) followed by pat dry.
d. adding HRP-conjugated anti-human IgA in to the wells followed by incubation for 3hoursat 4˚C and washing the wells thrice using wash buffer(1xPBS with 0.05% Tween-20, 150µl in each well) followed by pat dry.
e. adding TMB (Tetramethylbenzidine)/H2O2(Hydrogen peroxide) in to the wells followed by incubation for 30 minutesat room temperature in dark
f. adding 2N sulphuric acid in to the wells for arresting the reaction and measuring OD at 450nm using ELISA plate reader to obtain Total IgA (O.D450).
In accordance with the invention, in the method, the detection of total immunoglobulin isotope IgG is determined by sandwich ELISA using anti-human IgG antibodies comprises of following sequential steps.
a. coating wells in ELISA plate with the anti-human IgG, followed by incubation overnight at 40C and the next day washing thrice using wash buffer(1xPBS with 0.05% Tween-20, 150µl in each well) and pat dry
b. blocking wells with blocking buffer (1% BSA in 1X PBS, 25µl per well) for 3 hour at room temperature followed by washing the wells and pat dry
c. adding serum samples (1:1000 dilution) into the blocked wells followed by incubation overnight at 40C and washing the wells thrice using wash buffer(1xPBS with 0.05% Tween-20, 150µl in each well) followed by pat dry.
d. adding HRP-conjugated anti-human IgG in to the wells followed by incubation for 3hours at 4˚C and washing the wells thrice using wash buffer(1xPBS with 0.05% Tween-20, 150µl in each well) followed by pat dry.
e. adding TMB (Tetramethylbenzidine)/H2O2(Hydrogen peroxide) in to the wells followed by incubation for 30 minutes at room temperature in dark
f. adding 2N sulphuric acid in to the wells for arresting the reaction and measuring OD at 450nm using ELISA plate reader to obtain Total IgG (O.D450).

Although the invention has now been described in terms of certain preferred embodiments and exemplified with respect thereto, one skilled in art can readily appreciate that various modifications, changes, omissions and substitutions may be made without departing from the spirit thereof. It is intended therefore that the present invention be limited solely by the scope of the following claims.