Equine-RID Test

Company: Plasvacc

Active Ingredient(s): An immunodiffusion plate containing antiserum to equine IgG in agarose gel.

Equine-RID Test Indications

The product is used for the quantification of equine IgG by radial immunodiffusion.

Test Principles: Radial immunodiffusion is based on the diffusion of antigen from a circular well radial into a homogeneous gel containing specific antiserum for each particular antigen. A circle of precipitated antigen and antibody forms, and continues to grow until the equilibrium is reached. The diameters of the rings are a function of antigen concentration. After overnight incubation, the zone diameters of reference sera are plotted against the logarithm (base 10) of the antigen concentration. If equilibrium is reached, the reference sera zone diameters are squared and plotted against their concentration (linear). At intervals in between, a linear relationship does not occur. Unknown concentrations are measured by reference to the standard curve.

Test Procedure: A means of measuring five (5) microliters is required to do the test. Suitable pipettes may be supplied if necessary. The test will work with either plasma or serum.

1. Reagents:

A. Radial immunodiffusion plates contain specific antiserum in agarose gel, 0.1M phosphate buffer pH 7.0, 0.1% sodium azide as a bacteriostatic agent, 1 mcg/mL amphotericin B as an antifungal agent. The plates also contain 0.002M ethylenediaminetetraacetic acid. Store in a refrigerator at temperatures of (2 to 8°C).

B. Equine reference sera - (pooled equine serum at three (3) levels*). Contains sodium azide (0.1%) as a bacteriostatic agent. Store at refrigerator temperature.

2. Specimen Preparation and Handling:

A. Collect whole blood without anticoagulant and allow it to clot at room temperature.

B. Separate the serum by centrifugation at approximately 200 rcf within two (2) to three (3) hours after collection.

C. Plasma may be used, but nonspecific precipitation of fibrin may obscure the precipitation rings. In addition, liquid anticoagulants such as ACD fluid will dilute the specimen.

D. Caution: As noted above, 1B - the unknown specimens should be treated as infectious.

3. Procedure:

A. Materials Provided:

1. Three (3) radial immunodiffusion plates.

2. Reference sera 3 x 0.25 mis.

3. Directions for use.

B. Materials Required:

1. Blood collection tubes.

2. Centrifuge (200 rcf).

3. Microliter dispenser (5 microliters).

4. Reference sera (required if not provided in kit form).

5. Normal control sera (optional) - available separately.

6. Measuring device - calibrated in 0.1 mm increments.

7. Two cycle semi-logarithmic graph paper and/or linear graph paper.

C. General:

1. Do not overfill or underfill the wells. An improperly filled well yields erroneous results and the same specimen should be placed in another well. Overfilling with a five (5) microliter sample indicates that some gel shrinkage has occurred.

2. The reference serum zone diameters should be measured at the same time as the test sera. If a delay in the measurement is anticipated, allow sufficient intervals between filling the wells.

3. The time of filling each plate should be marked on the cover and if more than one (1) plate is filled, they should be read in order of filling.

4. Excess moisture is required to prevent drying. Replace each plate in its plastic bag and reseal it carefully before incubation.

5. The shrinkage of gel or oval shaped wells indicate drying and the plate should not be used.

6. If temperature fluctuations are anticipated, the plates in their bags may be incubated in an insulated container. Fluctuations in temperature may result in multiple precipitin ring formation.

7. The unused sections may be run at a later day if the plate has been stored at 2 to 8°C between incubations in its plastic bag. Check carefully for any evidence of drying.

8. Rough granulation of the gel indicates freezing, therefore the plates should be discarded.

D. Performance of Test:

1. Remove the plates from the refrigerator to room temperature (approximately 30 minutes) before filling the wells. Do not open the bag until it is ready for use.

2. If excess moisture is present, remove the plate from the bag and remove the cover until evaporation has dried the surface and the wells. Replace the cover until it is ready to be used.

3. For the best results, three (3) wells should be filled with reference sera for each plate. The location of each should be noted. Mix each vial of reference serum thoroughly.

4. Deliver the specimen to the well by placing the pipette tip at the bottom of the well. Allow the well to fill to the top of the agar surface. Avoid bubbles to ensure proper volume and diffusion of the sample. Visualization may be aided by placing the plate on a dark background. If practice is required, a used plate may be utilized.

5. More consistent results may be obtained when the wells are filled with a five (5) microliter pipette.

6. Mark the time of the completion on the plate cover and replace the cover.

7. Replace the plate in the bag and reseal it carefully.

8. Incubate the plates in an upright position on a flat surface at room temperature (20-24°C) for 16-20 hours for overnight readings and over 48 hours for end point readings. See C6 above.

E. Calibration:

1. Using the reference sera provided in the kits, determine their ring diameters to the nearest 0.1 mm.

2. Using 2 or 3 cycle semi-logarithmic graph paper, plot the concentration on the Y axis and the zone diameters on the linear or X axis for each protein for overnight readings.

3. Using regular graph paper, plot the concentration on the X axis and the zone diameters squared on the Y axis for each protein for end point readings.

4. Draw a straight line of “best fit” between the three (3) points. A curved line usually indicates that the incubation time and/or temperature should be reduced for overnight values. For valid results, a smooth curve should be fitted to the points and control sera included for additional verification.

F. Quality Control: For consistent results and a comparison of lot to lot, day to day, and week to week variations, a “normal” and abnormal serum should be included each day. The diameters and concentrations obtained can be charted to determine means and standard deviations. For the same specimen, an appropriate series of the wells on the same plate should yield diameters within 0.2 mm of one another. The control sera should be freshly thawed or reconstituted.

G. Reference Sera: All reference sera supplied have been calibrated from two (2) standard sera. The standard sera were calibrated against the appropriate purified proteins.

4. Results: Determine the concentration of each unknown of the specimen protein by reading its zone diameter on the reference curve and the corresponding concentration from the X axis. The zone diameter must be squared for end point calibration.

5. Interpretation of Results and Limitation of the Procedure:

A. When an unknown diameter exceeds that of the top standard, the specimen should be diluted with saline and rerun.

B. When an unknown diameter is smaller than that of the lowest standard, its concentration should be reported as “less than” the concentration of the reference serum. If available, “low level” radial immunodiffusion plates may be utilized.

C. Lack of a precipitin ring may be due to:

1. The sample not applied to the well.

2. A concentration too low to be detected by the method.

3. A concentration too high, resulting in the formation of soluble complexes, which are not precipitated.

D. The plates do not measure substitute colostrum sources of IgG from goats, sheep or cows.

6. Expected Values: The incidence of failure of passive transfer (FPT) of immunoglobulins has been estimated to be between 2.9 and 25%.^3,4,5 Partial passive transfer has been defined as immunoglobulin levels of 200 to 400 mg/dl. The total failure of passive transfer has less than 200 mg/dl.

The minimum level of IgG necessary to protect a foal from infection depends upon a number of factors, including the types of bacteria in the environment, management and stress factors and the colostral antibody titer against specific bacteria in the environment. Evidence suggests that foals should have IgG concentrations greater than 800 mg/dl.

The half-life of IgG from colostrum is 20 to 23 days^6,7 therefore serum immunoglobulin levels are at their lowest between one (1) to two (2) months of age.^8,9

These values are intended as a guideline - each laboratory should establish its own “normal” range. Values vary with age and should be established separately.

7. Performance Characteristics:

A. For investigational use only. The performance characteristics of the product have not been established.

Precaution(s): Store the plate upside down in a refrigerator.

Avoid jarring and freezing temperatures.

Discussion: Single radial immunodiffusion tests have evolved from the work of Fahey and McKelvey¹ and Mancini et al.² They are specific for the various proteins in serum or other fluids and depend upon the reaction of each protein with its specific antibody.

Immunoglobulin G (IgG) is one of the first line of defenses against encapsulated bacterial and streptococci. The majority of the newborns IgG is obtained from the dam's colostrum in the first 16 hours after birth, providing the foal nurses. This is called passive transfer. In passive transfer, the IgG from colostrum provides antibodies to infectious agents that the dam has been exposed to, or immunized against. The time it takes IgG to drop to half its original titer in mammals ranges from 20 to 30 days. The foal can start producing its own IgG in sufficient quantities after 30 to 80 days.

References

Available upon request.

Presentation: 24 wells.

CPN: 11280021