To determine the presence or absence of antigens or antibodies in a patient, many tests have been developed that use the interaction of antibodies and antigens. These tests require specificity and sensitivity. Specificity refers to the property of only binding to one antigen. Sensitivity means that the antibody can recognize and bind the antigen.
- Precipitation reactions depend on the interaction between antibodies and soluble antigens (acellular e.g. In the presence of electrolytes (NaCI), at a suitable temperature, pH and humidity
- These two insoluble reactants combine to form the precipitate, an insoluble product.
- Precipitate settles to bottom of tube in aqueous solution or appears as an opaque line (in gel).
- Antigen detection is sensitive to precipitation
- Precipitation tests can detect as little as 1g protein
- Precipitation, however, is less sensitive than other methods for the detection and measurement of antibodies.
- The following factors influence the amount of precipitate that is formed:
- The relative proportions of antibodies and antigens
- The Ab passion
- Both antibodies and antigens are complexes made up of amino acids. They also have positive and negatively polar groups that are distributed on their surfaces in certain but reciprocal patterns.
- Mixing antigen with corresponding antibody molecules results in electrical attraction and repellence. This causes the antigen and antibody molecules to be oriented according to their molecular forms, and electrical charges. Thus, an absolute fit (mold and cast), is achieved.
- Precipitation reactions are the reaction of the soluble antigens with IgG and IgM antibodies to form interlocking molecular aggregates known as lattices.
- Two distinct phases are involved in precipitation reactions. The first is when the antigens and antibodies quickly form small antigen-antibody combinations. This interaction takes place in seconds. Then, a slower reaction occurs, which can take minutes to hours. In this case, the antigen-antibody compounds form lattices that precipitate from the solution.
- Precipitation reactions can only occur when the antigen-antibody ratio is optimal.
Figure 1 shows that there is no precipitate when one component is too high. Separate solutions of antigen or antibody should be placed next to each other so that they can diffuse together. This is the best way to get the best ratio. The concentration of antibody and antigen in the equivalence zone is where there is complete precipitation.
Objectives of Ring Precipitation Test
- To detect antigen-antibody complexes.
- Describe the conditions under which antigen-antibody compounds precipitate out of solution.
- It is possible to detect antibodies in patient serum, which can be used for diagnosing disease.
Principle of Ring Precipitation Test
The precipitin ring test is a quick and qualitative method to determine the presence of antigen/antibody complexes. The precipitin curve principle states that when antigen and antibody are at the right ratio, they interact to form a cross-linked precipitate. The antiserum is added to a small-diameter test tube and carefully mixed with the antigen to form an upper layer. After four hours of incubation, a ring formed of precipitate at the contact point in the presence of antigen-antibody reactions. The concentration of antigen will determine the rate at which visible rings form.
Semi-quantitative results may be obtained by comparing experimental results with those derived using a standard curve using known amounts of antigen or antibody.
A series of dilutions are required to detect the precipitin reaction. Because both insufficient or excessive amounts of antigen can prevent the formation of visible precipitate, The presence of a thick layer of granulation between the antiserum solution and the antigen solution is a sign of an optimal antibody-antigen ratio.
- Saline (0.85%) NaCl
- Bovine globulin antiserum
- Normal bovine serum diluted to 1:25, 1:50, and 1:75 with physiological saline.
- 0.5ml pipettes
- Serological test tubes (8* 75mm)
- Serological test tube rack
Procedure of Ring Precipitation Test
- Three serological test tubes should be labeled according to the antigen concentration to be used (1.25, 1.50 and 1:75). The fourth tube can then serve as a saline test.
- Use a sterile 0.5-ml pipette to transfer 0.3 ml of each of the normal bovine serum dilutions into the appropriate labeled test tube.
- Use a clean, 0.5-ml pipette to transfer 0.3 ml saline to the control tube.
- All four tubes should be carefully covered with 0.3 ml bovine globulin-antiserum.
- To prevent sera from mixing, tilt the test tube and let the antiserum run down the side.
- All test tubes should be incubated for 30 minutes at 37°C.
- Examine all tubes to determine if there is a ring or precipitation at the interface. In the Lab Report, indicate whether a ring is present.
- Record the amount of antigen dilution that caused the most precipitation. This is the best antibody:antigen ratio.
Results Interpretation of Ring Precipitation Test
- Positive Test: The development of a white ring near the intersection of antiserum solution and antigen solution is a positive sign.
- Negative Result: The absence of a ring formation.
Uses of Ring Precipitation Test
- Used in C-reactive protein (CRP).
- Used for Lancefield grouping of β-haemolytic streptococci.
- Ascoli’s thermoprecipitin test used for anthrax diagnosis.
- WALSHE JM, LACHMANN PJ, ORREGO-MATTE H. A ring precipitin test in chronic liver disease: a study of the false positive. Immunology. 1961 Jul;4(3):232-5. PMID: 13782928; PMCID: PMC1424043.
- Deshmukh, Amol & Dighe, Pravin & Tiwari, Kundan & Garud, Supriya. (2020). PRECIPITATION REACTIONS IN IMMUNOLOGY. EUROPEAN JOURNAL OF PHARMACEUTICAL AND MEDICAL RESEARCH. 7. 214-216.
- Boyden, A. (1963). Precipitin Testing and Classification. Systematic Zoology, 12(1), 1. doi:10.2307/2411889