KIT DIAGNOSTIC

EMIRITA WULAN P.
G0011083

 Recent

advances in molecular biology and more generally in

biotechnology may help to resolve these problems through the

development of field diagnostic kits.
The ideal field diagnostic kit should, of course, be sensitive and specific

(like any other laboratory technique) but should also be easy to use by
unskilled persons and resistant to rough climatic conditions.

DEVELOPMENT OF FIELD KITS

Probes
Polymerase chain reaction
Kits with monoclonal antibodies
Protein expression by a vector

PCR
The polymerase chain reaction (PCR) is a scientific technique in

molecular biology to amplify a single or a few copies of a piece of DNA

across several orders of magnitude, generating thousands to millions of
copies of a particular DNA sequence.
was developed in 1984 by the American biochemist, Kary Mullis.

The basic PCR principle: a chain reaction: One DNA molecule is used to produce two

copies, then four, then eight and so forth.

This continuous doubling is accomplished by specific proteins known as polymerases,

enzymes that are able to string together individual DNA building blocks to form long
molecular strands.
To do their job polymerases require a supply of DNA building blocks, i.e. the nucleotides

consisting of the four bases adenine (A), thymine (T), cytosine (C) and guanine (G).
They also need a small fragment of DNA, known as the primer, to which they attach the

building blocks as well as a longer DNA molecule to serve as a template for constructing the
new strand.

 three major steps involved in the PCR technique: denaturation,

annealing, and extension.

ELISA
ELISA

is a rapid test used for detecting and

quantifying antibodies or antigens against viruses,
bacteria and other materials.
In ELISA technology, the solid phase consists of a 96well polystyrene plate, although other materials can be
used. The function of the solid phase is to immobilize
either antigens or antibodies in the sample, as they
bind to the solid phase. After incubation, the plates are
washed to remove any unbound material. In some
assays the conjugate is then added to the plate and
allowed to incubate.

 The conjugate consists of either an antigen or antibody that has been

labeled with an enzyme. Depending upon the assay format, the

immunologically reactive portion of the conjugate binds with either the
solid phase or the sample. The enzyme portion of the conjugate enables
detection.
The plates are washed again and an enzyme substrate (hydrogen

peroxide and a chromogen) is added and allowed to incubate. Color

develops in the presence of bound enzyme and the optical density is
read with an ELISA plate reader.

ELISAs are divided into several formats.

Indirect

Direct

Competitive

IHC
Immunohistochemistry (or IHC) is a method for demonstrating the

presence and location of proteins in tissue sections.

Though less sensitive quantitatively than immunoassays such as

Western blotting or ELISA, it enables the observation of processes in

the context of intact tissue. This is especially useful for assessing the
progression and treatment of diseases such as cancer. In general, the
information gained from IHC combined with microscopy literally
provides a big picture that can help make sense of data obtained
using other methods.

Western Blotting
Western blotting uses specific antibodies to identify proteins that have

been separated based on size by gel electrophoresis.

The immunoassay uses a membrane made of nitrocellulose or PVDF

(polyvinylidene fluoride).
The gel is placed next to the membrane and application of an electrical

current induces the proteins to migrate from the gel to the membrane.
The membrane can then be further processed with antibodies specific
for the target of interest, and visualized using secondary antibodies and
detection reagents.

THANK YOU