HLA, HLA TYPING IMMUNOGLOBULINS ESTIMATION

HLA = Human Leucocyte Antigen system

HLA forms part of the Major Histocompatibiblity Complex (MHC) Found on the short arm of chromosome 6 MHC antigens are integral to the normal functioning of the immune response. Essential role of HLA antigens lies in the control of self recognition and defence against micro-organisms and surveillance.

 HLA comprises two classes: Class I Class II . Class I - A,B,C most significant (other loci eg. E,F,G,H etc are not so important in transplantation) . Expressed on most nucleated cells . Have soluble form in plasma . Are adsorbed onto platelets (some antigens more readily than others) Erythrocytes will adsorb some Class I antigens viz. Bg blood group system (B7,A28, B57.) HLA B most polymorphic system followed by A and then C 45Kd glycoprotein comprising three heavy chain domains

 non-covalently associated with beta-2-microglobulin (coded chromosome 15) which plays an important role in the structural support of the heavy chains. Class I molecules are assembled within the cell and ultimately sit on the cell surface with a section inserted into the lipid bilayer of the cell membrane and a short cytoplasmic tail where they present antigen in the form of peptide to cytotoxic T (CD8+) cells

 HLA Class II - five loci DR, DQ, DP, DM and DO HLA DR, DQ, DP most significant Expressed on B lymphocytes, activated T lymphocytes, macrophages, endothelial cells ie immune competent cells. Comprise 2 chains encoded by HLA genes, alpha and beta each with 2 domains. Hypervariable region is in the beta 1 domain

 HLA Class II present peptide in the cleft to helper T (CD4+) cells. Thus Class II presentation involves the helper-function of setting up a general immune reaction involving cytokine, cellular and humoral defence. The role of Class II in initiating a general immune response is why they only need to be present on immunologically active cells.

TYPING METHODS
SEROLOGY - used to be the gold standard. Now being replaced by molecular techniques CELLULAR - rarely used now. Orginally used for Class II typing MOLECULAR - fast becoming the method of choice. Many laboratories test of choice.

SEROLOGY
Complement Dependent Cytotoxicity (CDC) Viable peripheral blood lymphocytes are obtained by discontinous density gradient centrifugation using Ficoll / Tryosil or Ficoll / Sodium Metrizoate at a density of 1.077 at 19 - 22C. Microlymphocytotoxic test: 3 stages

Microlymphocyototoxic test
1. Viable lymphocytes are incubated with HLA specific antibodies. If the specific antigen is present on the cell the antibody is bound. 2. Rabbit serum as a source of complement is added, incubate. If antibody is bound to the HLA antigen on the cell surface it activates the complement which damages the cell membrane making it permeable to vital stains.

Microlymphocyototoxic test
Results are visualised by adding dye usually a fluorochrome eg Ethidium Bromide. If the reaction has taken place the EB enters the cell and binds to the DNA.

Microlymphocytotoxicity test
Test is left for 10 minutes and then read using an inverted fluorescient microscope. A mixture of T and B lymphocytes can be used for HLA Class I typing. B lymphocytes are required for HLA Class II typing by serology. (Normal population 8590% T and 10-15% B cells) This can be achieved using a number of methods.

Microlymphocytotoxicity test
In the past neuraminidase treated sheep red blood cell rosetting and nylon wool have been used. Immunomagnetic bead separation is the current method of choice.

Advantages Easily performed does not require expensive equipment. Takes around three hours to perform Low level resolution, with good antisera reliable results Disadvantages Requires large volumes of blood Requires viable lymphocytes Difficult to find good antisera for rarer antigens in different populations

Cellular typing
Not / Rarely used by laboratories these days. Requires panels of homozygous typing cells. Cell culture method therefore takes a long time. Labour intensive involves use of radioisotopes.

Molecular
All commonly used molecular methods require good quality genomic DNA. There are numerous methods for extraction of DNA from whole blood. Manual / commercial kits All methods rely on DNA extraction from the nucleated cells following cell lysis and protein digestion

Molecular Methods
Most methods currently used have a PCR element within the technique. PCR Three steps per cycle denaturation, annealing and extension

Molecular Methods
SSP (Sequence Specific Priming) Can be used for HLA Class I and II typing using a panel of primer pairs either for low to medium resolution whereby primers amplify groups of alleles or high resolution whereby primer pairs amplify specific alleles. Each PCR reaction takes place in a separate tube therefore the number of tubes depends on the level of resolution. Each tube also contains a pair of primers for part of the human growth hormone gene as an internal control.

Molecular Methods
Electrophoresis is used following amplification. PCR product is run out on an agarose gel containing ethidium bromide. Each product moves according to its size and is compared to a molecular weight marker. Results are visualised using 312nm UV transillumination and recorded either by video imaging or polaroid photograghy.

Molecular methods
PCR SSOP ( Sequence Specific Oligonucleotide Probes) Dot blot in house method usually whereby one labels ones own probes with Digoxigenin Reverse dot blot normally commercial where specific oligonucleotide probes are attached to a nylon membrane.

Pros and Cons

Advantages Does not require viable cells Samples do not have to arrive in the lab the day they are taken PCR SSOP good for batch testing Can be semi automated Disadvantages Requires good quality DNA Require a degree of redundancy within the primers used Sequence of alleles must be known.

Molecular Methods
Sequence Based Typing (SBT) DNA sequencing is the determination of the sequence of a gene and thus is the highest resolution possible. Sequence based typing involves PCR amplification of the gene of interest eg HLA DRB1 followed by determination of the base sequence. The sequence is then compared with a database of DRB1 gene sequences to find comparable sequences and assign alleles. This method also allows for detection on new alleles.

Measurement of Ig
Immunodiffusion precipitation reactions Agglutination reactions Immunoelectrophoresis ELISA

1) Precipitation reaction
When a soluble antigen having at least

two or more antibody binding sites react with the corresponding antibody in suitable concentration, it leads to the formation of large Ag-Ab complexes which are visible to the naked eye as a precipitate. Formation of an Ag-Ab lattice depends on the valency of both antibody and antigen.

2) Agglutination Reactions
When antigen is of particulate nature, its

reaction with antibodies leads to the formation of visible clumps called agglutination. Ig M antibodies are more efficient in causing such reaction

(a) Haemagglutination /slide agglutination

Hemagglutination

involves agglutination of RBCs. Zeta potential IgM is more effective than IgG in agglutinating red blood cells.

(b) AGGLUTINATION INHIBITION absence of agglutination is diagnostic of antigen

Applications of agglutination inhibition reactions: Pregnancy test to determine whether an individual is using certain types of illegal drugs, such as cocaine or heroin. Viral infections in premarital testing to determine the immune status of women with respect to rubella virus.

(c) Bacterial gglutination/Tube agglutination

The agglutinin titre of an antiserum can be used

to diagnose a bacterial infection. E.g: typhoid, typhus fever, streptococcus and brucella infections.

1)RADIAL IMMUNODIFFUSION (Mancini)ASSAY

Ag

1)RADIAL IMMUNODIFFUSION ASSAY

d [ Ag ]

Radial Immunodiffusion
To quantitate: Ig levels in serum AFP Screening sera for Ab to influenza virus.
Ab in gel
Ag Ag Ag Ag

Diameter2

Ag Concentration

3)TURBIDIMETRIC ASSAY
Measures turbidity or cloudiness of a solution by

measuring the amount of light PASSING THROUGH the solution. Soluble antigen and antibody join and once they join in sufficient amounts precipitate, results in cloudiness. The more cloudy the solution, the less light can pass through.

a) Single diffusion in single dimension (Oudin procedure)

Here concentration gradient is established for only a single reactant. A Quantitative technique based on this principle: RADIAL IMMUNODIFFUSION

b) Double diffusion in single dimension

c) Double diffusion in two dimension

Ouchterlony - Identity
Precipitation appears as

a continuous line in the form of an arc between the two outer wells and the center well. There are no spurs at the angle and this type of reaction is termed a band of identity.

Ouchterlony Partial Identity

This indicates that the two

antigenic materials in wells 1 and 2 are related, but that the material in well 1 possesses an antigenic specificity not possessed by the material in well 2. Such a reaction with spur formation indicates partial identity

Ouchterlony Non-Identity
If the material in wells 1

and 2 do not possess common antigens and the antiserum in well 3 possesses specificities for both materials, the reaction will appear as two crossed lines.

2) Immunoelectrophoresis
Combines electrophoresis with immunometric detection. Electrophoresis provides separation Immunoprecipitation provides detection.

Immunoelectrophoresis
Mixture of Antigens, Evaluation of specificity of antisera, Human gammopathies

Specimen

Shape and position of arc are characteristic of separated protein

Two dimensional immunoelectrophoresis/ Crossed Immunoelectrophoresis (CRIE)

3) Western Blotting
Purpose: Detects levels of antibodies being produced

4) Dot blotting
This method is similar to western blotting but it

bypasses the electrophoretic separation step. Protein sample to be analysed is applied to a membrane surface as a small dot and dried. The membrane is then exposed to a labeled Ab specific for the test Ag contained in the dotted protein. After washing, bound Ab is detected with a photometric or chemiluminescent detection system.