Diagnostic Kit

Rafi Amanda Rezkia A.

Umi Hani Vismayanti L.
20151030

Diagnostic kit?

Kit = equipment needed for a specific

purpose
Diagnostic = Theidentificationof the
nature of an illness or other problem
Diagnostic kit = in-vitro diagnostic
(IVD)

Kategories of IVD

Clinical laboratory testing

Near-patient testing
Self-testing

Type of Tests Based on the

Purpose of Test

Clinical Tests
Are used to examine sample specimens of individual

patients for diagnosis, prevention, or treatment of

genetic related disorders.

Research Tests
Are conducted in laboratories and research centers

to study and understand genetic conditions or to

develop clinical level tests.

Investigative and Recreational Tests

Are considered valuable tests, but are those that

have not gained either scientific

acceptance in the medical community.

validity

or

Basic Knowledge in
Designing Diagnostic Kit

Antigen-antibody
Interaction

Antigen

Marked
by
immune
system as a foreign
substance.
Can bind with the product
of
adaptive
immune
system (immunoglobulin).
Has
unique
structure
recognized by antibody
epitope
(antigenic
determinant).
Immunogen?

Designing Antigen

Based on genomic analysis.

Region

of difference (RD) 1-4 found in

Mycobacterium tuberculosis H37Rv but absent in
vaccine strain of M. bovis var BCG ESAT-6 and
CFP-10 genes (located in RD1) reported as
dominant antigents recognized by T-cells &
consider as virulence factors in M. tuberculosis
target for recombinant protein for diagnosis
purpose.

Amplification using a specific PCR primer

cloning in a vector protein expression
purification.

Antibody/Immunoglobulin

Y-shape protein produced by plasma

cells that is used by the immune
system to identify and neutralize
pathogens such as bacteria and
viruses.
The amino acid sequence in the tips
of the "Y" varies greatly among
different antibodies (variable region)
specificity for binding antigen.
Antibodies are divided into five
major classes, IgM, IgG, IgA, IgD,
and IgE, based on their constant (Fc)
region
structure
and
immune
function.

The variable region is subdivided into:

Hypervariable (HV): directly contact with
antigen's
surface

complementarity
determining regions (CDRs).
Framework (FR) regions: serves as a scaffold
to hold the HV regions in position to contact
antigen.

Polyclonal Antibodies

Produced by plasma cells.

Recognize multiple epitopes on the surface of
antigen.
Conformational changes may not influence all
epitopes to the same degree.
Useful for immunoprecipitation for complex
antigens because the antibodies can bind more
than one antigen molecule forming a large
precipitating lattice.
Species selection: goat/rabbit.

Monoclonal Antibodies

Produced by a single B lymphocyte.

Was first recognized in sera of patients with
multiple myeloma in which clonal expansion of
malignant plasma cells produce high levels of an
identical antibody resulting in a monoclonal
gammopathy.
In the mid-1970s, Khler and Milstein devised the
technique for generating monoclonal antibodies of
a desired specificity They fused splenic B cells
with myeloma cells with the resulting immortal
hybridomas, each producing a unique MAb.

Have high specificity recognize a

single specific epitope less crossreactivity.
Influenced
by
conformational
determinants, and may not bind the
same protein in a denatured state.
Conformational changes association
with other proteins, post-translational
modification, temperature, pH, salt
concentration, and fixation.
Species selection: rat/mouse.

Application

ELISPO
T

RIDT

Assay Development and Validation Pathway

Benefit of using diagnostic

kit
provide objective information about a
persons health

Types of diagnostic kit

product

Reagents
Analytical instruments
Accessory products

The perfect kit

A kit should contain everything needed

to allow testing
the reagents should be absolutely
stable under a wide range of
temperature conditions
the manual describing the use of the
kit should be foolproof
the kit should be validated

The perfect kit (2)

external quality assessment should be

included in the kit package
data on the relationship of kit results to
those from other assays should be
included
attention should be given to ensuring
that all equipment
used in association with the kit is
calibrated

The perfect kit (3)

Criteria for ideal diagnostic

kit

FDA approval

High sensitivity and specificity be

compared with gold standard

Assessment and Evaluation

1. Performance
Characteristic

To distinguish infected from uninfected

individuals.
Sensitivity (%): probability that a truly

infected individual will test positive.

Specificity (%): probability that a truly
uninfected individual will test negative.

Determined
against
a
standard gold standard.

reference

Positive predictive value (PPV) (%):

probability that those testing positive by
the test are truly infected.
Negative predictive value (NPV) (%):
probability that those testing negative by
the test are truly uninfected.
PPV depend on sensitivity and prevalence
of infection.

Reproducibility
assessment of the extent to which the

same tester achieves the same results on

repeated testing of the same samples, or
the extent to which different testers
achieve the same results on the same
samples.
is measured by the percentage of times
the same results are obtained when the
test is used repeatedly on the same
specimens.

2. Operational
Characteristics

Time taken to perform the test.

Technical simplicity or ease of use.
User acceptability.
Stability of the test under user
conditions.

Some gold Standard Assays to Determine the

Diagnostic Sensitivity and Specificity of Antigen
Test Kits

Some gold Standard Assays to Determine

the Diagnostic Sensitivity and Specificity
of Antibody Test Kits

Challenges of making
diagnostic kit

Import Dependency
Limited funding
Regulatory Issues
Lack of Training and Education