Determination of Blood Insulin Concentration by Enzyme Linked

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Determination of blood Insulinconcentration by enzyme

linked immunosorbent assay(ELISA)


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Content

Biological substance: insulinDiseases associated with insulin

disturbances

Cases

Method of measurement: ELISADefinition

Principle

Equipments

Types

Principle of insulin ELISA and the

procedure.


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Insulin

Human insulin is a peptide hormonecomposed of 51 amino acids.

It is produced in the islets of

Langerhans in the pancreas.

It is a hormone central to regulating

carbohydrate and fat metabolism in

the body.


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Diseases associated withinsulin disturbances

Elevated insulin levels are seen with:1. Insulinomas

2. Insulin resistance, such as appears in

obesity, type 2 diabetes and metabolicsyndrome.

Decreased insulin levels are seen with:

1. Diabetes

2. Pancreatic diseases such as chronicpancreatitis (including cystic fibrosis)and pancreatic cancer


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Cases CASE I:A 50-year-old female presents to your clinic

with complaints ofexcessive thirst, fluidintake, and urination. She denies any urinarytract infection symptoms. She reports nomedical problems, but has not seen a doctorin many years. On examination she is anobese female in no acute distress. Herphysical exam is otherwise normal. The urineanalysis revealed high glucose level, and aserum random blood sugar level was 320mg/dl.What is the most likely diagnosis?A case oftype II DM (might be insulinresistance state).


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CASE II

An 82-year-old white woman presented to her

primary care physician with history ofepisodic confusion and somnolence. Theepisodes occurred typically in the morning,

just after waking. They lasted minutes and

were relieved when she ate her breakfast orhad juice. As she was waiting for check-out,she developed confusion, a capillary bloodglucose test was performed, and she wasnoted to have a plasma glucose level of 28mg/dl. She was given juice and hersymptoms resolved after a few moments.

What is the most likely diagnosis?

A case ofinsulinomas (islet cell tumors of thepancreas).


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Principle:

Antigen:a high molecular weight (>5000) substance

recognized by the immune system of anorganism as foreign.

The organism reacts to antigen by producinga specific antibody.

Hapten:a low molecular weight (


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Antibody (immunoglobulin):a large Y-shaped protein

used by the immune system to identify andneutralize foreign objects such as bacteriaand viruses.

recognizes a unique part of the foreign

target, termed an antigen. ELISA:is based on an antigen-antibody

(immunological) reaction

which is followed by an indicator reaction, toallow the enzyme activity bound in antigen-antibody complex. This occurs in thepresence of a substrate, giving a specificcolor. This color can be photometricallydetermined.


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Equipments:

Microtiter plate

Microplate sealing tape

Automatic pipettes


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ELISA washer

ELISA reader


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Types ofELISA:

Indirect ELISA

Sandwich ELISA

Competitive ELISA


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Indirect ELISA

Used to measure

antibody levels.

The antibodyreacts with specific

antigen attached to

the solid phase.


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Indirect ELISA


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SandwichELISA

The double

antibody method.

The unknown

antigen reacts withspecific antibody

attached to the

solid phase.


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Sandwich ELISA


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CompetitiveELISA

Enzyme- labeled

antigen and the

unknown amount

of the antigen inthe sample react

with a specific

antibody attached

to a solid phase.


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Principle of the insulin ELISA

The Insulin ELISA Kit is an enzyme-linked immunosorbent assay based onthe sandwich principle.

The microtiterwells (solid phase) are

coated with a monoclonal antibodydirected towards a unique antigenic siteon the Insulin molecule.

Samples and standards are pipetted into

these wells, along with the enzymeconjugate (monoclonal antibody againsthuman insulin, which has horseradishperoxidase (HRP) enzyme covalentlylinked to it).


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During the incubation, the insulin

antigen binds to the immobilizedcapture antibodies. At the same time

the conjugate antibody is binding to

the insulin present in the well forming

the sandwich.

The amount ofbound HRP complex is

proportional to the concentration of

Insulin in the sample.


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Afterwashing to remove unbound

materials, a substrate for HRP is

added.

The substrate is converted to a

colored compound by the action ofHRP.

The intensity of colourdeveloped is

proportional to the concentration ofInsulin in the patient sample.


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Assay Procedure


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Calculation of Results

Calculate the absorbance for eachstandards and patient samples using

the ELISA reader.

Standard Optical Units (450 nm)

Standard 0 (0 ulU/ml)0.03

Standard 1 {6.25 ulU/ml)0.07

Standard 2 (12.5 ulU/ml)0.14

Standard 3 (25 ulU/ml) 0.35

Standard 4 (50 ulU/ml)0.83

Standard 5 (100 ylU/ml) 2.05


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A standard curve isconstructed by

plotting the meanabsorbance obtainedfrom each standardagainst itsconcentration with

absorbance value onthe vertical(Y) axisand concentration onthe horizontal (X)axis.

Using the meanabsorbance value foreach sampledetermine thecorresponding

concentration fromthe standard curve.


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Determination of Blood Insulin Concentration by Enzyme Linked

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