Multiplex Testing in Immunoassay - Dr. Budi Santosa, M.si, Med

CURRICULUM VITAE

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Nama : Dr. Budi Santosa, MSi.Med Pekerjaan : Dosen Unit Kerja : Universitas Muhammadiyah Semarang Program Studi Analis Kesehatan Pendidikan :

Organisasi :

1. 2. 3. 4.

1990 : 1990 2000 20 00 : 2009 20 09 : 2013 201 3: UNDIP

AAK AA K Sur Surak akar arta ta FKM UND FKM UNDIP IP Biomed Biom edik ik UND UNDIP IP Kedokter Kedok teran/ an/Kes Keseha ehatan tan

PATELKI (Ketua DPW JATENG : 20132017)

IMMUNOLOGI ASSAY: Multiplex Immunoassay Budi Santosa Seminar Ilmiah Rakernas PATELKI Swiss Bell Hotel SKA Pekanbaru Pekan baru 22 22 Mei 2015

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An immunoassay is a test that uses antibody and antibody  and antigen antigen complexes  complexes as a means of generating a measurable result.. result

Immuno & assay 

immune response r esponse that causes “Immuno Immuno” ” refers to an immune the body to generate antibodies,

and    “assay” refers to a test. Thus, an immunoassay is a test that utilizes immunocomplexing immunocomplexing when antibodies and antigens are brought together

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Immunoassays  are different from other Immunoassays are types of laboratory tests, such as colorimetric tests, tests, because they use antibody:antigen complexes to generate a signal that can be measured.

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An antibody is a protein that is produced by the body in response to an “invading” (foreign) substance. Antibodies are produced as part of the body’s immune response to protect itself.

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Antibodies possess high a) specificity and  b) affinity for a specific antigen. It is the specific binding of an antibody to an antigen that allows the detection of analytes  by a variety of immunoassay methods.

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Antibodies (Ab) are a type of protein called immunoglobulins. The most common one is immunoglobulin G (IgG). IgG is a protein composed of two main structural and functional regions

Struktur Antibodi

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An antigen is the substance that the body is trying to “fight off” (eliminate or reduce) by mounting an immune response. Some immunoassays test for antigens directly. For example, the drug is the antigen that binds to the antibody.

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An analyte is anything measured by a laboratory test. In immunoassay testing, the analyte may be either an antibody, or an antigen.

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Immunoassays utilize one or more select antibodies to detect analytes of interest. The analytes being measured may be those that are: a) naturally present  in the body (such as a thyroid hormone), b) the body produces but are not typically present (such as a cancer antigen), c) do not naturally occur in the body (such as an abused drug).

IMMUNOASSAY

ELISA

MULTIPLEX -

TEST

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Enzyme-linked immunosorbent assay Name suggests three components ◦

Antibody 

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Solid phase (sorbent) 

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Allows for specific detection of analyte of interest Allows one to wash away all the material that is not specifically captured

Enzymatic amplification 

Allows you to turn a little capture into a visible color change that can be quantified using an absorbance plate reader

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Measure antibody levels (allergies, vaccines) Detect viruses (hepatitis, HIV, venereal diseases) Detect hormonal changes (pregnancy) Detect circulatory inflammatory markers (cytokines)

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Sensitivity Quantitative Reproducible Kit format

Relative sensitivities of tests (approx) Usual operating range [Ab] or [Ag] precipitation immunoelectrophoresis double/radial diffusion immunofluorescence

10 g/ml - 1 mg/ml

0.1 - 10 g/ml

ELISA (colour) (chemiluminescence)

0.1 - 10 ng/ml 0.01 - 10 ng/ml

radioimmunoassay

0.01 - 10 ng/ml

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Different Types ◦ ◦ ◦

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Indirect Sandwich Competitive

Can Be Used To Detect Both Antibody and Antigen Very Sensitive, pg/mL (Ex. 10 pg/mL)

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A substrate for this enzyme is then added. Often, this substrate changes colour upon reaction with the enzyme. The colour change shows that secondary antibody has bound to primary antibody, which strongly implies that the donor has had an immune reaction to the test antigen.

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The higher the concentration of the primary antibody that was present in the serum, the stronger the colour change. Often a spectrometer is used to give quantitative values for colour strength

Sandwich ELISA •

The ELISA plate is coated with Antibody to detect specific antigen

Sandwich ELISA

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Less is more. More antigen in your sample will mean more antibody competed away, which will lead to less signal

One step competi ti ve format 

In the one step competitive format (see Figure) both the labeled antigen reagent (Ag*) and the unlabeled specimen (or test sample analyte) compete for a limited amount of antibody.

Two step competi ti ve f ormat

a. Indirect ILISA

b. Sanwich ILISA

c. Competitive ILISA

RESUME

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Result of Elisa reader Std 1

Std 2

Dcs

PGE2

LPS

LPS + -5

-6

-7

-8

Neg Ctrl

6.125

0.331

0.275

0.099

0.094

0.315

0.168

0.268

0.289

0.319

0.098

3.0625

0.183

0.18

0.1

0.095

0.31

0.172

0.268

0.285

0.297

0.095

1.53125

0.155

0.136

0.106

0.099

0.286

0.179

0.263

0.263

0.266

0.104

0.765625

0.139

0.13

0.105

0.105

0.322

0.205

0.278

0.298

0.279

0.102

0.382813

0.127

0.12

0.111

0.106

0.324

0.204

0.309

0.353

0.292

0.12

0.191406

0.118

0.112

0.112

0.12

0.31

0.204

0.326

0.308

0.324

0.108

0.116

0.11

0.045

0.042

0.052

0.052

0.053

0.051

0.042

0.042

0.123

0.123

0.044

0.052

0.051

0.052

0.054

0.052

0.052

0.053

Sample Standard Curve 0.5 0.45    ) 0.4   m0.35   n    0    9 0.3    4    (   e 0.25   c   n   a 0.2    b   r   o 0.15    b    A

0.1

absorbance

0.05

Log. (absorbance)

0 0.1

1

y = -0.0583Ln(x) + 0.3858 R2 = 0.9919 10

Concentration (ug/mL)

100

MULTIPLEX IMUNOASSAY

What is Multiplex Assay?

Laxman B, Morris DS, Yu  J et al. (2008)).

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Principles of Bead-Based Multiplex Immunoassays

 Assay Principle

Sample is added to microspheres and analite is capture

Fluorescent tagged detection antibody is added

Laser detect both bead dyes and tagged detection antibody

Dual Laser: 1 klasification bead 2. Derived sgnal to proportion with analyte

A magnet capture analyte and hold bead magnetic Two dioda illuminate the beads 1. LED for detection 2. Derived sgnal to

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Simultaneous detection of up to 100 analytes Get results in 3-4 hours Low sample volume requirement saves precious sample Better sensitivity and dynamic range compared to ELISA Easy-to-use software for instrument control data visualization and data management Flexible ordering options: preconfigured readyto-use kits or create-your-own custom assays World class support from Bio-Rad, the first company to partner with Luminex to deliver xMAP assays, instruments, and software

1

Allergy Testing

8 Genotyping

2

Autoimmune

9 Infectious Disease

3

Cancer Markers

10

Isotyping

4

Cardiac Markers

11

Metabolic Markers

5

Cytokine

12

Tissue Typing

6

EndocrineACTH (h),

13

Kinase Phosphorylated

Adiponectin

Protein

(h,m), Amylin (m) (rt) (h), CPeptide (h), Calcitonin (h), CRF Linco 7

Gene Expression

14

Transcription FactorsNuclear Receptors

1.Biomarker validation; 2.Standardisation of immunoassay design and quality control (calibration and quantification); 3.Availability, stability, specificity and cross– reactivity of reagents; 4. Assay automation and the use of validated algorithms for transformation of raw data into diagnostic results

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 Efficiency workflow and staffing ◦

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Limiting hands on time Single operator

Shorter Turn around time ◦

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Multiplex : All target 1 – 6 h Singleplex 

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Bacterial : 2-3 days Viral : EIA 1-2 h single plex 5-8 Parasite : 3 – 5 days

Preventive drug resistance Cost still a big challenge ◦

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Value of shorter hospitalization Earlier initiate therapy Avoiding unnecessary treatment viral infection