Agglutination Part 2

Latex agglutination In latex agglutination procedures, Ag molecules can be bound to the surface of latex beads. If Ab is present in the test specimen, the Ag will combine with the Ab and form visible aggregates.

Latex agglutination Latex particles can be coated with Ab, and in the presence of Ag can form visible aggregates.

Reverse Indirect agglutination Involves the adherence of Ab to inert particles which can then be used to detect the presence of Ag. Example – latex particle coated with Ab (known) + serum looking for (unknown) Ag. If Ag present, then you get visible agglutination.

Reverse Indirect agglutination Numerous kits are available for rapid identification of antigens on infectious agents Examples: Staphylococcus aureus Neisseria maningetidis Mycoplasma pneumoniae Such tests used for organisms that are difficult to grow or when rapid identification is required

Reverse Indirect agglutination Used also to measure levels of therapeutic drugs, hormones, plasma proteins

Hemagglutination The agglutination of red blood cells by either direct agglutination or indirect agglutination Direct agg. Ag is an intrinsic component of RBC Indirect agg. Soluble Ags are adsorbed to the RBC

Hemagglutination There are 3 ways in which Ags may be bound to RBCs: Spontaneous adsorption of Ags by RBCs Covalent binding using chemical links Tannic acid treatment

Hemagglutination Y + ↔ Qualitative agglutination test Ag or Ab

Hemagglutination Quantitative agglutination test Titer Prozone 1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512 1/1024 Pos. Neg. Titer 64 8 512 <2 32 128 4 Patient 1 2 3 5 6 7

Indirect Hemagglutination Definition - agglutination test done with a soluble antigen coated onto a particle Y + ↔

Agglutination inhibition / Hemagglutination inhibition Involves interference by Ag or Ab with an Ag-Ab reaction which would have resulted in agglutination, if interference had not occurred. The technique is called hemagglutination inhibition if the particle in the reaction is a rbc.

Agglutination inhibition - pos Tube containing Ab (known) Patient has Ag (unknown) and combines with Ab. Not visible. Latex bead coated with known Ag is added. It has nothing to attach to – no visible reaction or agglutination inhibition is positive.

Agglutination inhibition - neg Tube containing Ab (known). Patient serum does not contain Ag (unknown), therefore no combination. Latex bead coated with known Ag directed to known Ab coated to tube. Visible agglutination, therefore agglutination inhibition is negative

Agglutination inhibition

Hemagglutination Inhibition Definition - test based on the inhibition of agglutination due to competition with a soluble Ag Y Y + + ↔ Test Y Patient’s sample

Coagglutination Coagglutination (CoA) is similar to the Latex Agglutination technique for detecting antigen Protein A, a uniformly distributed cell wall component of Staphylococcus aureus, is able to bind to the Fc region of most IgG isotype antibodies leaving the Fab region free to interact with antigens present in the applied specimens. The visible agglutination of the S. aureus particles indicates the antigen-antibody reaction.

Coagglutination

Coagglutination These particles exhibit greater stability than Latex particles However, bacteria are not colored & therefore reactions are often difficult to read. Such tests are highly specific, but the sensitivity is less than latex particles method

Viral Hemagglutination Many viruses have nonserological hemagglutinating properties ِThey can agglutinate RBCs in the absence of Ab (nonimmune agglutination)

Viral Hemagglutination Hemagglutination (HA) can be used to determine titers of certain viruses. Mammalian reoviruses agglutinate erythrocytes through interactions between the viral surface protein sigma 1 and carbohydrate groups attached to proteins on erythrocyte membranes. Visually, this interaction creates a shield of erythrocytes in a round-bottom 96 well plate (well F1). In the absence of virus, the erythrocytes form a button in the well (well F12).

Viral Hemagglutination

Slide indirect Hemagglutination test Exercise 4

Principle The Waaler-Rose (WR) reagent is a suspension of stabilized sheep red cells sensitized with anti-sheep rabbit IgG. The WR test reagent sensivity has been adjusted to detect a minimum of 6 IU/mL of rheumatoid factors according with the WHO International Standard without previous sample dilution.

Sample Use fresh serum obtained by centrifugation of clotted blood. The sample may be stored at +2 oC to +8 oC for 48 hours before performing the test. For longer periods of time the serum must be frozen. Hemolytic, lipemic or contaminated sera must be discarded

Reagents Waaler Rose: Suspension of solubilized sheep erythrocytes sensitized with rabbit IgG anti-sheep erythrocytes Positive Control Negative Control

Preparation of reagents Shake the Reagent 1 (red cells) before use. After that it must be uniform and without visible clumping. The sensitivity of the test depends of the drop volume (50 μl). Do not use droppers than those provided and hold the dropper perpendicular to the slide surface. The reagent and controls have to be stored at +2 oC to +8 oC. Do not freeze. In these conditions the components will remain stable until the expiry date printed on the label.

Procedure Before using the kit, allow the components to reach room temperature. Gently shake the reagent to disperse the particles. Check the reagent against the positive and negative controls in the same way as the undiluted serum.

1. Qualitative Determination Serum 1 drop (50 μl) + Reagent 1 drop Place a drop of UNDILUTED serum onto a circle of the slide. Add a drop of the Reagent 1 (red cells) next to the drop of serum. Mix both drops spreading them over the full surface of the circle. Let the slide to stay on a flat surface for two minutes. After this time twist the slide 45 degrees once and let again to stay for one minute. Read the presence or absence of visible agglutination in this period of time. Unespecific agglutination could appear if the test is read later than this time.

Interpretation of Results Examine macroscopically the presence3 or absence of visible agglutination immediately avoiding any movement or lifting of the slide during the observation. The presence of agglutination indicates a content of RF in the sample equal or greater than 8 IU/ml. The lack of agglutination indicates a RF level lower than 8 IU/ml in the sample

Semi-Quantitative method This test is performed in the same way as the qualitative test but by using previous dilution of the serum sample in saline (NaCl 9 g/l),

Semi-Quantitative method

Calculations 8 X RF titer = IU/ml The approximate RF concentration in the patient sample is calculated as follows: 8 X RF titer = IU/ml