immuno....seminar fin

8/9/2019 Immuno....Seminar Fin

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INTRODUCTION TO IMMUNOSENSORS

ANTIBODIES TO HEAVY METALS

Siva P.Gurunathan

Wilson G.Anandaraj

[email protected]


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HEAVY METAL POLLUTION *

Heavy metals are toxic and persistent environmental

contaminants.

Metals often persist in the environment for long periods of

time bound to soils or sediments.

changes in weather, in the pH of the soil or water, or in other

combinations of environmental factors can mobilize bound

metals and greatly increase their availability and

effective toxicity.

So the heavy metal analysis in different environment is

needed

to measure the level of metals.

* Antibodies to Heavy Metals: Isolation, Characterization, and Incorporation intoMicro plate-BasedAssays and Immunosensors: Diane A. Blake et al.


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ENVIRONMENTAL MONITORING

INSTRUMENTAL ANALYSISVs IMMUNO SENSORS APPLICATION*

Instrumental analysis are expensive , the time required for

analysis can be prolonged.

Antibody- based techniques are alternative approaches for

metal ion analyses.

Attractive to local and governmental agencies.

Quick , inexpensive , simple to perform , portable ,

Sensitive & Selective.

Can reduce analysis costs by 50 % or more.

*Antibody-based sensors for heavy metal ions Diane A. Blake et al.


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ANTIBODY BASED IMMUNOSENSOR

The availability of antibodies to heavy metals would

permit the construction of immunosensors for

environmental monitoring works.

Purified mouse monoclonal antibodies withspecificities for chelated cadmium, lead, cobalt,

uranium and Chromium were available from previous

studies.

The metal ion binding properties and metal-ion

specificities of these antibodies have been described(Blake et al., 1996,1998a, 2001; Khosraviani et al.,

2000).


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PREPARATION OF THE IMMUNOGEN

CHOOSING THE

CORRECT CHELATOR

SYNTHESIS OF

PROTEIN CONJUGATES


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CHOOSING THE CORRECT CHELATOR

Why we need chelator ?

Heavy metals do not elicit immune response have relatively

open structures that facilitate diffusion-controlled binding

of the metal to the chelator.

When a heavy metal was added to a chelator covalently

conjugated to a protein, the resulting metal chelate complex

comprised an epitope that was immunogenic and could

elicit an immune response required for rapid immune assay.

Metal chelate complexes by themselves are too small to

stimulate the immune system.

Examples of Chelators :

EDTA , DTPA , Cyclohexyl DTPA , 2,9 dicarboxyl 1 , 10 phenanthroline.


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CHELATORS Generation of

Antibodies with

Bifunctional Derivatives ofEDTA

Bifunctional Derivatives of DTPA

Bifunctional Derivatives of

Cyclohexyl- DTPA

Bifunctional Derivatives of

2,9 dicarboxyl 1 , 10 phenanthroline


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a] Bifunctional Derivatives ofEDTA , b ] Bifunctional Derivatives of DTPA

C ] Bifunctional Derivatives of Cyclohexyl- DTPA

d ] Bifunctional Derivatives of 2,9 dicarboxyl 1 , 10 phenanthroline

CHELATORS


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SYNTHESIS OF PROTEIN CONJUGATES

PROTEIN+ CHELATOR +METAL

METALCHELTORPROTEIN CONJUGATES

Act as Immunogens

Contin.


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METALCHELTORPROTEIN CONJUGATES


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Preparation of protein - chelate complexes was based on

Chakrabartis report.

2 mg of protein (KLH or OVA) was dissolved in 3 mL of 100 mM

boric acid [or HEPES buffer](pH 9.0) and then mixed with 1 mg

of iso thio cyanobenzyl EDTA overnight.

During this time, The isothiocyanate on the bifunctionalchelate reacted with unprotonated amino groups on the

carrier protein to form a stable thioureido group.

Before the conjugation with EDTA, the pH of the solution

as adjusted to 7.0 using a desalting column.

The reaction rate increased with increasing pH .it was

maintained at 9 with 10 M KOH.

Keyhole Limpet Hemocyanin [KLH] and Bovine Serum Albumin

are used as the Protein Carrier.

Contin.

Contin.


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Keyhole Limpet Hemocyanin has been the carrier protein of

choice for immunization.

It stimulates T-cell dependent responses and often used ascarrier for low molecular weight antigens.

BSA is structurally dissimilar to KLH ,readily soluble ,

metal free form.

The conjugates are washed with the metal free HEPES Buffer .

The metal solution was added drop wise with gentle stirring

to the purified ITCBEprotein conjugate; the pH was

adjusted to 7.0 to avoid precipitation.

The binding reaction proceeded for 3 h at roomtemperature.

The metal- Chelate -Protein conjugate was separated from

low molecular weight.

Reaction products by ultra filtration or Size Exclusion

Chromatography.

Contin.


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ANTIBODY PRODUCTION


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MONO CLONAL ANTIBODY PRODUCTION


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IMMUNIZATION OF MICE


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Mice were injected intraperitoneally at 2 week intervals with Metal -EDTA-

KLH conjugate emulsified in adjuvant.

The fourth injection was given 7 days after the third injection. Three days

after the fourth injection, the reactivity of sera toward 10 M MetalEDTA

and 10 M metal free EDTA were analyzed with a KinExA or ELISA system.

Four days after the fourth injection, spleen cells were fused with myeloma cells

using polyethylene glycol 1500.

The fused cells were cultured in six 96 well flat-bottomed micro culture plates for

14 days.

Hybridoma cells were cloned by colony formation using methylcellulose semisolid

medium.

Ascitic fluids were produced in mice by injecting hybridoma cells.

The purity of the antibody was confirmed by HPLC (high-performance liquid

chromatography)


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SOLID PHASE PREPARATION

Antigen-coated Sepharose beads were prepared as follows.

One milliliter of NHS-activated Sepharose suspension was washed with

0.1N HCl and then neutralized with phosphate buffered saline (PBS)

buffer (pH 7.4).

Metal-EDTA-Protein conjugate solution (1 mg of protein/mL) was added

to Sepharose beads and mixed gently overnight.

Finally, Sepharose beads were blocked against nonspecific binding withBSA solution (1 mg/mL) by mixing over 2 h.


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To immobilize EDTA Protein conjugate to polystyrene micro plates 50 L

of the conjugate solution (0.01 mg/ml) was added to each well and kept overnight

at 4 C.

After the plate was washed with water, 50 L of 1 mM metal solution was added tomake the Metal -EDTA complex on the solid phase.

After washing with PBS containing 0.1% (w/v) Tween-20 (T-PBS buffer), 200 L of

T-PBS with 10% fetal bovine serum (FBS) was added to block

for 1 h at room temperature.

The blocking solution was discarded, and then each well was washed

with T-PBS.

Immunoassay Procedure for ELISA

Contin..


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Each hybridoma supernatant (25 L) was allowed to bind Pb-EDTA-OVA

on the plate for 30 min.

After discarding the supernatant and washing with T-PBS for three times, the

second antibody (goat anti-mouse IgG-alkaline phosphatase conjugate wasallowed to bind for 30 min.

Binding of the second (alkaline phosphatase-labeled) antibody was detected

using an alkaline phosphatase substrate kit .

Alkaline phosphatase activity was measured using p-nitrophenyl phosphate asthe substrate and monitoring absorbance at 410 nm with a plate reader.

Contin..


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(a) Two step competitive ELISA.

1) The wells of a microtitre plate were coated

with a metalchelate -BSA conjugate andnonspecific binding sites were blocked with

BSA.

2) The sample (or standard) was mixed with

chelator and a known concentration of

primary antibody, and the mixture was added

to the micro well where the immobilized andthe soluble antigens compete for primary

antibody binding.

3) Unbound antibody was washed away

4) Bound primary antibody was detected with

an HRP- conjugated anti-species secondaryantibody.

5) Reaction color is developed by addition of

HRP substrate.


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(b) One-step competitive ELISA.

(1) The wells of a microtitre plate were coated with primary

antibody to the metalchelate complex and non-specific

binding sites were blocked with BSA.

(2) The sample (containing metalchelate complex) was

spiked with a known concentration of HRP conjugated

metalchelate complex, and this mixture was allowed to

bind to the immobilized primary antibody. Enzyme-linked

and unconjugated metalchelate complexes competed for

binding to the immobilized antibody.

(3) Excess unbound antigen is washed away.

(4) Reaction color is developed by addition of HRP substrat


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(1) Sample (or standard) containing metal-chelate complex

was mixed with known concentrations of primary

antibody, and the mixture was allowed to come to

equilibrium.

(2) The equilibrated antibodyantigen solution was then

passed rapidly over the metalchelate complex

immobilized on the surface of beads packed into a

capillary bed.

(3) Only those antibodies without metal-chelate complex

in their binding sites were available to bind to the

immobilized antigen; unbound antibody-antigen

complex was washed away.

(4) Bound primary antibodies captured in the capillary

bed were detected by using a fluorescently-labeled

anti-species secondary antibody.

(c) Sensor Assay.


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