Immune Assays

Precipitin Assays:

Diffusion PPTE Technology

1. Interfacial Test or Ring Test
- First quantitative immunological assay
- Can be used to detect either Ag or Ab present in a system, can be used to find point of equivalence.

Double Diffusion PPTE Technology
2. Ouchterlony Assay
- diffusion of both Ag and Ab in semisolid media (agar, agarose), or on a wetted matrix (sheets of cellulose acetate soaked in buffer).
- line of ppte forms at point of equivalence of the 2 advancing fronts of Ag and Ab.

Advantages of Ouchterlony Test
1. See more than 1 Ag
2. Can identify Ags
a. line of identity
b. nonidentity
c. partial identity
1. Time
2. Sensitivity

3. Immunoelectrophoresis (IEP)
- combines double diffusion with electrophoresis
- 1st separate Ag by electrophoresis
- cut trough // to electrophoretic field
- add Ab to trough and let both Ag and Ab diffuse
- can run known Ag for identification of Ag

Incorporated Gel Technology
Either Ag or Ab is incorporated as part of matrix.

4. Single Radial Immunodiffusion (Mancini Test).
- either Ag or Ab is incorporated into gel
- the other is placed in a well and allowed to diffuse into gel
- a ppte ring forms, the size depends on Ag/Ab concentration
- know conc. run to develop standard curve (measure diameter)
- sensitive to µg/ml Ag
- used to study immunodifficiency syndromes or multiple myelomas

5. Rocket Immunoelectrophoresis
- Laurell in 1966, has for many assays replaced SRID, gives accuracy with speed of electrophoresis
- Ag electrophoresis into gel containing Ab
- Ag will ppte with the height of the rocket prop. to [Ag]. Area under peak prop to [Ag].
- run standards of known conc. to develop standard curve
- advantage. takes 3 hours, not days.
Why does this work?
- pH 8.6 no charge on Ig, they don't migrate

6. Two-Dimensional Immunoelectrophoresis (XIE)
- also developed by Laurell
- 1st separate Ags by electrophoresis as in [IEP]
- 2nd electrophoresis first gel into 2nd incorporated gel, not diffused in as in IEP
- can see complexity of IR
- can add an intermediate gel to identify peaks

Agglutination Assays
- depend on agglutination of Ag gives a semiquantitative assay of Ab levels or of specificity depending on how one sets up the assay
- dependent on cross linking and ppte by polyvalent Ab
- quantify by dilution of Ab. The highest dilution giving a +ve reaction is defined as the titer.
- specificity defined as a +ve or -ve reaction with a specific Ag, or agglutination inhibition assays, use soluble hapten to inhibit agglutination

7. Bacterial Agglutination
- is there Ab to bacteria?
mix Ab + bacteria on slide, look for agglutination
Widal Test - Typhoid fever
Whooping Cough - B. pertussis

8. Hemagglutination Assays
- ABO Typing
- Rh
Coombs' Test - increase ppte by adding a second Ab to assay. Rh test developed this way.

9. Passive Hemagglutination
- use RBC as carrier of Ag of interest
- treat RBCs with tannic acid or chomic chloride to "sensitize" surface, then add Ag to bind to RBC
- Beads: RBC's can be replaced with latex or polystyrene beads. Come in many sizes and colours. Bind protein of interest, block with BSA or skim milk protein, etc. Carry out agglutination assay.
-Inhibition of Agglutination: Can measure soluble Ag as a competitive inhibitor of agglutination. (Home Preg. Test, Factor VIII)
Sensitivity - 0.05 µg protein

Second Ab or Second Receptor Assays
In some of these assays it is possible to do a single or direct assay, using 1 Ab or marker, these are much less sensitive, 2 component system is the choice in most cases.

10. Complement Fixation:
- one of the first multicomponent assays
- can measure either Ab or Ag levels
2 step assay
1. Ag-Ab interaction

- dilutions of Ag or Ab make assay quantitative
- control is preimmune serum
- add C' (Guinea Pig)
- incubate 4·C for 18 hrs. C' has short life at room temperature

In tubes where an Ag-Ab reaction takes place, the C' will be bound to that complex. In tubes where no Ag-Ab reaction takes place, C' is free to bind to other Ag-Ab complexes.

2. Detection System
- detection assay usually involves sheep RBC (SRBC) and antibody to SRBCs (AbSRBC)
- add detection system to the incubated tubes and incubate a further 30-60 min at 37·C

Reading the assay
1. Control in preimmune serum.
No Ab to Ag therefore no Ag - Ab interaction and C' is free to bind SRBC - AbSRBC complex leading to lysis of SRBC
2. In test samples
Ag + Ab form Ag - Ab complex that can then bind C' Ag - Ab - C' complex leaves no free C' therefore, when AbSRBC - SRBC complex forms there is NO C' TO LYSE CELLS.

11. RadioImmune Assay (RIA)
Put immune assays on the map because it could measure nano to pico gram levels of Ag. Therefore can measure hormones, growth factors, cytokines, etc., which could only be measured in bioassays prior to RIA. Developed by Berson & Yadow 1960 - insulin in plasma
Competitive Binding Assay: form standard curve by adding fixed concentrations of Ag-I125 and Ab. To this is added known concentrations of cold Ag to compete with the labeled Ag. The ppte that forms from the Ag-Ab interaction (can use a second Ab to increase ppte.) is collected by centrifugation and the counts in the pellet and in the supernatent are determined. Can be used to measure Ag in any solution by preparing dilutions of solution (blood, urine, etc.) and comparing inhibition to a known concentration of cold Ag.
- want to work in linear part of binding curve

- sensitivity
- automated
- modified for solid substrates.
- can bind Ag to well and use I125 Ab or Staph Protein A
- do not need to purify test sample

I125, has a short half life, therefore must make Ag-I125 fresh. Costly in time, need pure Ag to I125 label. I125 not nice.

12. Enzyme-Linked ImmunoSorbent Assay (ELISA)
- has become the Gold Standard
- high sensitivity (pico gram range)
- automated
- no radioactive Ag
- done in 96 well plate made of special plastic, which binds Ag very tightly
- detection assay based on enzyme-linked second Ab or Protein A
- enzyme is peroxidase, alkaline phosphatase urease, substrates are chromogens. Substrate is colourless yields a
coloured end product.
Colour, which is proportional to [Ag-Ab], is read on a Spectraphotometer.

13. Western Blot
- combines SDS-PAGE with ELISA technology
- not a quantitative assay but used to identify Ag
STEP 2 - electroblot to polymer sheet nitrocellulose (Immobilon P)
STEP 3 - block sheet surface with protein (BSA, Casiene)
STEP 4 - Ag - Ab reaction
STEP 5 - enzyme-linked secondary Ab
STEP 6 - chromogen

14. Immunohistochemistry
- use Ab to identify the presence and distribution of Ag in tissue
- technology similar to ELISA or Western Blot, but it's done on tissue sections
STEP 1 - fix, embed and cut section
STEP 2 - add nonimmune serum from same animal species as secondary Ab as a blocking agent
STEP 3 - add specific immune serum
STEP 4 - add secondary Ab with detection system

- fluorescent label
- enzyme
- radioactive tag
- biotin-avadin
STEP 5- view slides

15. Fluorescent Activated Cell Sorter (FACS)
- can be used to identify, quantify and sort cells according to surface membrane Ags using fluorescent labeled Ab.
- add Ab to Ag of interest, the Ab is fl-conjugated. Can use >1 Ab with different fl tags.
fluorescein absorbs blue (490) emits yellow-green (517)
rhodamine absorbs yellow-green (515) emits red (546)

16. Jerne Plaque Assay (Hemolytic Plaque Assay)
- developed by Jerne in the U.S. and Ingraham and Bussard in France(1960's)
- measures Ab secretion by plasma cells. Can identify plasma cell secreting Ab of interest.

1. Immunize mouse with SRBCs.
2. Wait 4 days, remove spleen and prepare a single cell population of spleen cells.
3. Mix with SRBCs, add to warm agar, allow to solidify. [SRBC] should lead to uniform layer of SRBCs. Incubate for a few hours.
4. Add C', incubate at 37·C.

Assay can be used with other Ags using RBC as passive carriers of the Ag.

17. Cutaneous Anaphylaxis (P-K Test)
- early 1981 by Prausnitz and Kustner
- demonstrated transfer of allergic reaction in serum
- serum from test animal is injected under skin of responder animal
- next day inject Ag at same site
- if +ve get instantaneous wheel and flare reaction
- IgE arms mast cells at injection site
- Ag leads to mast cell activation
immediate type hypersensitivity

18. Mantoux (Delayed type hypersensitivity)
TB test. involves cells not IgE
Ag sensitive. host, 4 days reaction
Passive transfer cells not serum.

19. Lymphokine Production
- can assay for products of immune response (cytokines, growth factors, etc.) to identify if an immune response has been initiated or to identify the mechanism through which signaling of the response occurs. These were once done by bioassay,e.g. MIF Migration Inhibition Factor inhibits macrophage migration, assay for macrophage migration in vitro ± MIF. Now most factors are measured by ELISA.

20. Allograft Rejection
- skin from donor placed over skin lesion, hold in place 9 days with bandage.
- rapid rejection ¸ 14 days; slow rejection ¸ weeks

21. Graft vs Host
- foreign lymphocytes placed in host can attack host tissue in non-self histocompatible host.
- early sign is enlargement of spleen
- graft vs host measured by spleen index.
SI ¸ 1.0 = no reaction
SI _ 1.3 = GVHR

22. Mixed Lymphocyte Reaction (MLR)
- cells from 2 individuals which differ in MHC will react to each other stimulating cell replication, which can be assayed by 3H-T incorporation
- take 4-5 days after cells mixed, add 3H-T for 18 hrs. One way MLR.

1. Stimulator cells don't grow (mitomycin c, irrad.)
2. Responder cells grow in response to stimulated. cells
3. Control - mix of cells from same strain

23. Cell Mediated Cytotoxicity (CMC)
- measure cytotoxic response between lymphocytes that differ in MHC
- modified from MLR
1. Killer cells activated as in MLR, stimulator cells (MitC) are added to responder cells for 5 days activation of responders.
2. Target cells are made from the same cell population as stimulator cells by inducing growth with a mitogen (lectin like ConA). Label cells with 51CrO4. Measure cell killing by CrO4 release.