Complement Fixation Test (CFT)

This test is based upon two properties of the complement viz:

a. Complent combines with all antigen-antibody complexes whether or not it is required for that reaction
b. Complement is needed in immunolytic reaction.

Test system

It contains an antigen and a serum suspected to be having antibody to that antigen. The serum is heat treated prior to the test to destroy its complement. Complement Is added in measured quantity to this system. This complement is the form of guinea pig serum which is considered a rich source of complement. The test system is incubated.

Indicator system

To test system, after incubation, is added the indicator system which consists of sheep
RBCs and antibody to sheep RBCs (haemolysin) and another incubation is allowed.
If there is specific antibody in the test system, it will bind to antigen and to this complex the complement will also get fixed. Hence, no complement will be available to combine with indicator system which though contains RBCs and their specific antibody, cannot undergo haemolysis unless complement gets attached. Absence of haemolysis shall indicated positive test or presence of specific antibody in the serum which has been added in the test system. Erythrocytes lysis is obtained in negative test.

DISINFECTION AND STERILIZATION

•    Sterilization is the best destruction or com removal_of all forms of micro organisms.
•    Disinfection is the destruction of many microorganisms but usually the b spores.
•    Antisepsis is the destruction or inhibition of microorganisms in living tissues thereby limiting or preventing the harmful effect of infection.
•    Astatic Agent  would only inhibit the growth of microorganisms (bacteriostatic, fungistatic, sporostatic).
•    Acidal agent would kill the microorganism (bactericidal. virucidal, fungicidal)
•    Sterilants are the chemicals which under controlled conditions can kill sporinQ bacteria.
 

Immunology:

The branch of life science which deals with immune reaction is known as immunology.

Components of Immune System:

The immune system consists of a network of diverse organs and tissue which vary structurally as well as functionally from each other. These organs remain spreaded throughout the body. Basically, immune system is a complex network of lymphoid organs, tissues and cells.

These lym­phoid organs can be categorized under three types depending upon their functional aspects:

i.  Primary lymphoid organ.

ii. Secondary lymphoid organ.

iii.Tertiary lymphoid organ.

White blood cells or leukocytes are the basic cell types which help to give rise to different types of cells which participate in the development of immune response . WBC are classified into granulocytes and agranulocytes depending on the presence or absence of granules in the cyto­plasm.

Agranular leukocytes are of two types, viz., lymphocytes and monocytes. Lymphocytes play pivotal role in producing defensive molecules of immune system. Out of all leukocytes, only lymphocytes possess the quality of diversity, specificity, memory and self-non self recognition as various important aspects of immune response.

Other cell types remain as accessory one; help to activate lymphocytes, to generate various immune effector cells, to increase the rate of anti­gen clearance 

All cells of the immune system have their origin in the bone marrow 

myeloid (neutrophils, basophils, eosinpophils, macrophages and dendritic cells) 

lymphoid (B lymphocyte, T lymphocyte and Natural Killer) cells .

The myeloid progenitor (stem) cell in the bone marrow gives rise to erythrocytes, platelets, neutrophils, monocytes/macrophages and dendritic cells whereas the lymphoid progenitor (stem) cell gives rise to the NK, T cells and B cells. 

For T cell development the precursor T cells must migrate to the thymus where they undergo differentiation into two distinct types of T cells, the CD4+ T helper cell and the CD8+ pre-cytotoxic T cell. 

Two types of T helper cells are produced in the thymus the TH1 cells, which help the CD8+ pre-cytotoxic cells to differentiate into cytotoxic T cells, and TH2 cells, which help B cells, differentiate into plasma cells, which secrete antibodies. 

Function of the immune system is self/non-self discrimination. 

This ability to distinguish between self and non-self is necessary to protect the organism from invading pathogens and to eliminate modified or altered cells (e.g. malignant cells). 

Since pathogens may replicate intracellularly (viruses and some bacteria and parasites) or extracellularly (most bacteria, fungi and parasites), different components of the immune system have evolved to protect against these different types of pathogens.

Immunofluorescence

This is precipitation or complement fixation tests. The technique can detect proteins at concentrations of around 1 µg protein per ml body fluid. Major disadvantage with this technique is frequent occurrence of nonspecific fluorescence in the tissues and other material.
The fluorescent dyes commonly used are fluorescein isothocyanate (FITC). These dyes exhibit fluorescence by absorbing UV light between 290 and 495 nm and emitting longer wavelength coloured light of 525 nm which gives shining appearance (fluorescence) to protein labelled with dye. Blue green (apple green) fluorescence is seen with FITC and orange red with rhodamine.

Enzyme Immunoassays

These are commonly called as enzyme linked immunosorbent assays or EL1SA. It is a simple and versatile technique which is as sensitive as radioimmunoassays. It is now the
technique for the detection of antigens, antibodies, hormones, toxins and viruses.

Identification of organisms by immunofluorescence

Type of agent         Examples

Bacterial            Neisseria gonorrhoeae, H. influenzae ,Strept pyogenes, Treponema pallidum
Viral                  Herpesvirus, Rabiesvirus, Epstein-Barr virus
Mycotic             Candida albicans

Enzymatic activity results in a colour change which can be assessed visibly or quantified in a simple spectrophotometer.

Immunoglobulin (Ig)

Immunoglobulins are glycoprotein molecules that are produced by plasma cells in response to an immunogen and which function as antibodies. The immunoglobulins derive their name from the finding that they migrate with globular proteins when antibody-containing serum is placed in an electrical field

FUNCTION
1. Immunoglobulins bind specifically to one or a few closely related antigens. Each immunoglobulin actually binds to a specific antigenic determinant. Antigen binding by antibodies is the primary function of antibodies and can result in protection of the host.

2. The significant biological effects are a consequence of secondary "effector functions" of antibodies.Phagocytic cells, lymphocytes, platelets, mast cells, and basophils have receptors that bind immunoglobulins. This binding can activate the cells to perform some function. Some immunoglobulins also bind to receptors on placental trophoblasts, which results in transfer of the immunoglobulin across the placenta. As a result, the transferred maternal antibodies provide immunity to the fetus and newborn.

STRUCTURE OF IMMUNOGLOBULINS

The basic structure of the immunoglobulins is illustrated in figure 2. Although different immunoglobulins can differ structurally, they all are built from the same basic units.

A. Heavy and Light Chains

All immunoglobulins have a four chain structure as their basic unit. They are composed of two identical light chains (23kD) and two identical heavy chains (50-70kD)

B. Disulfide bonds

1. Inter-chain disulfide bonds - The heavy and light chains and the two heavy chains are held together by inter-chain disulfide bonds and by non-covalent interactions The number of inter-chain disulfide bonds varies among different immunoglobulin molecules.

2. Intra-chain disulfide binds - Within each of the polypeptide chains there are also intra-chain disulfide bonds.

C. Variable (V) and Constant (C) Regions

When the amino acid sequences of many different heavy chains and light chains were compared, it became clear that both the heavy and light chain could be divided into two regions based on variability in the amino acid sequences. These are the:

1. Light Chain - VL (110 amino acids) and CL (110 amino acids)

2. Heavy Chain - VH (110 amino acids) and CH (330-440 amino acids)\(x = {-b \pm \sqrt{b^2-4ac} \over 2a}\)h the arms of the antibody molecule forms a Y. It is called the hinge region because there is some flexibility in the molecule at this point.

E. Domains

Three dimensional images of the immunoglobulin molecule show that it is not straight as depicted in figure 2A. Rather, it is folded into globular regions each of which contains an intra-chain disulfide bond (figure 2B-D). These regions are called domains.

1. Light Chain Domains - VL and CL

2. Heavy Chain Domains - VH, CH1 - CH3 (or CH4)

F. Oligosaccharides

Carbohydrates are attached to the CH2 domain in most immunoglobulins. However, in some cases carbohydrates may also be attached at other locations. 

IMMUNOGLOBULIN FRAGMENTS: STRUCTURE/FUNCTION RELATIONSHIPS

Immunoglobulin fragments produced by proteolytic digestion –

A.  Fab 
Digestion with papain breaks the immunoglobulin molecule in the hinge region before the H-H inter-chain disulfide bond Figure 6. This results in the formation of two identical fragments that contain the light chain and the VH and CH1 domains of the heavy chain.

Antigen binding – These fragments are  called the Fab fragments because they contained the antigen binding sites of the antibody. Each Fab fragment is monovalent whereas the original molecule was divalent. The combining site of the antibody is created by both VH and VL. 

B. Fc 
Digestion with papain also produces a fragment that contains the remainder of the two heavy chains each containing a CH2 and CH3 domain. This fragment was called Fc because it was easily crystallized.

Effector functions – The effector functions of immunoglobulins are mediated by this part of the molecule. Different functions are mediated by the different domains in this fragment . 

Treatment of immunoglobulins with pepsin results in cleavage of the heavy chain after the H-H inter-chain disulfide bonds resulting in a fragment that contains both antigen binding sites . This fragment is called F(ab’)2because it is divalent. The Fc region of the molecule is digested into small peptides by pepsin. The F(ab’)2binds antigen but it does not mediate the effector functions of antibodies.
 

Method of Sterilization for common items

Autoclaving :  Animal cages, Sugar tubes, Lab. Coats, Cotton , Filters, Instruments Culture media, Rubber, Gloves , Stopper, Tubing, Slides,  Syringe and Wax needles , Test tubes, Enamel metal trays ,Wire baskets, Wood, Tongue depressor, Applicator, Endodontic instruments, Orthodontic pliers , Orthodontic kits, Saliva ejector, Handpieces Cavitron heads, Steel burs, Steel tumbler, Hand instruments    

Hot air oven

Beakers, Flasks, Petri dish, Slides, Syringes, Test tubes, Glycerine, Needles ,Oil, Paper Saliva ejector, Matrix Band

Ethylene oxide

Fabric, Bedding, Blanket, Clothing, Matteresses, Pillows, Disposable instruments , Instruments, Blades, Knives, Scalpels, Scissors ,Talcum powder, Books, Cups, plates , Plastics., Flask, Petridish, Tubes, Tubing, Rubber , catheters, Drains, Gloves ,Special items - Bronchoscope, Cystoscope, Heart lung machine

Glutaraldehyde

Orthodontic kits, Orthodontic pliers , Steel burrs, 3 in 1 syringe tips ,Cystoscope ,Endoscope

Filtration

Antibiotics, Serum, Vaccines