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Fundamentals of In-House Serologic Kits
Fundamentals of In-House Serologic Kits
• Mechanisms: These kits primarily utilize ELISA (microwell or membrane-based), immunomigration, immunochromatography, and agglutination.
• Mechanisms: These kits primarily utilize ELISA (microwell or membrane-based), immunomigration, immunochromatography, and agglutination.
• Targets: They measure either antigens (direct detection of the agent) or antibodies (measuring the immune response or protection levels).
• Targets: They measure either antigens (direct detection of the agent) or antibodies (measuring the immune response or protection levels).
• Hormone Testing: Serologic formats are also used to measure various hormone levels.
• Hormone Testing: Serologic formats are also used to measure various hormone levels.
• Sample Types: Depending on the test, requirements may include plasma, serum, whole blood, feces, or saliva.
• Sample Types: Depending on the test, requirements may include plasma, serum, whole blood, feces, or saliva.
🐈Feline Viral Infections
🐈Feline Viral Infections
• Feline Leukemia Virus (FeLV):
• Feline Leukemia Virus (FeLV):
◦ Detection: Kits identify the soluble p27 gag protein produced during viremia.
◦ Detection: Kits identify the soluble p27 gag protein produced during viremia.
◦ Timing: Detectable antigen typically appears within 28–30 days post-infection.
◦ Timing: Detectable antigen typically appears within 28–30 days post-infection.
◦ Reliability: Testing serum, plasma, or whole blood is more reliable than saliva or tears.
◦ Reliability: Testing serum, plasma, or whole blood is more reliable than saliva or tears.
◦ Interpretation: While negative results are highly reliable, false positives can occur in low-incidence populations; thus, single positive results should not dictate clinical decisions without confirmation.
◦ Interpretation: While negative results are highly reliable, false positives can occur in low-incidence populations; thus, single positive results should not dictate clinical decisions without confirmation.
◦ PCR Advantage: Real-time PCR can identify "proviral DNA positive" cats that are antigen-negative and unlikely to shed virus but are still risky for blood transfusions.
◦ PCR Advantage: Real-time PCR can identify "proviral DNA positive" cats that are antigen-negative and unlikely to shed virus but are still risky for blood transfusions.
• Feline Immunodeficiency Virus (FIV):
• Feline Immunodeficiency Virus (FIV):
◦ Detection: Because antigen levels are low, these kits detect anti-FIV antibodies.
◦ Detection: Because antigen levels are low, these kits detect anti-FIV antibodies.
◦ Window Period: Antibodies usually develop within 60 days, though this is variable.
◦ Window Period: Antibodies usually develop within 60 days, though this is variable.
◦ Vaccination Interference: Current in-house kits cannot distinguish between antibodies from natural infection and those from vaccination.
◦ Vaccination Interference: Current in-house kits cannot distinguish between antibodies from natural infection and those from vaccination.
◦ Kittens: Positive results in kittens under 6 months may reflect maternally derived antibodies; they should be retested after 6 months of age.
◦ Kittens: Positive results in kittens under 6 months may reflect maternally derived antibodies; they should be retested after 6 months of age.
🐕Canine Viral Infections
🐕Canine Viral Infections
• Canine Parvovirus (CPV):
• Canine Parvovirus (CPV):
◦ Antigen Detection: Uses feces; however, shedding only lasts 7–10 days and may be missed if clinical signs appear late.
◦ Antigen Detection: Uses feces; however, shedding only lasts 7–10 days and may be missed if clinical signs appear late.
◦ Limitations: False negatives can occur due to blood in feces or the formation of antigen-antibody complexes; sensitivity may be lower for the CPV2c strain.
◦ Limitations: False negatives can occur due to blood in feces or the formation of antigen-antibody complexes; sensitivity may be lower for the CPV2c strain.
◦ Antibody Detection: Used to evaluate the need for revaccination or the presence of maternal antibodies.
◦ Antibody Detection: Used to evaluate the need for revaccination or the presence of maternal antibodies.
◦ Feline Use: While not licensed for cats, these kits can detect feline panleukopenia virus in feces.
◦ Feline Use: While not licensed for cats, these kits can detect feline panleukopenia virus in feces.
• Canine Distemper Virus (CDV):
• Canine Distemper Virus (CDV):
◦ Antibody Testing: Semiquantitative ELISA for IgG is used to evaluate revaccination needs and maternal antibody levels in puppies.
◦ Antibody Testing: Semiquantitative ELISA for IgG is used to evaluate revaccination needs and maternal antibody levels in puppies.
◦ Antigen Testing: Immunochromatography-based assays are best performed using conjunctival swabs but are not currently standard in-house kits.
◦ Antigen Testing: Immunochromatography-based assays are best performed using conjunctival swabs but are not currently standard in-house kits.
Tickborne and Bacterial Diseases
Tickborne and Bacterial Diseases
• Borrelia burgdorferi (Lyme Disease):
• Borrelia burgdorferi (Lyme Disease):
◦ C6 Peptide Technology: Uses a synthetic peptide based on a conserved protein to minimize cross-reactivity.
◦ C6 Peptide Technology: Uses a synthetic peptide based on a conserved protein to minimize cross-reactivity.
◦ Differentiation: Assays using the C6 protein can distinguish between vaccine-induced antibodies and natural infection.
◦ Differentiation: Assays using the C6 protein can distinguish between vaccine-induced antibodies and natural infection.
◦ Application: Validated for dogs, cats, and horses.
◦ Application: Validated for dogs, cats, and horses.
• Ehrlichia canis:
• Ehrlichia canis:
◦ Detection: Qualitative or semiquantitative ELISA for antibodies in serum, plasma, or whole blood.
◦ Detection: Qualitative or semiquantitative ELISA for antibodies in serum, plasma, or whole blood.
◦ Limitation: Long-lived antibodies mean a positive test does not distinguish between current illness and past exposure.
◦ Limitation: Long-lived antibodies mean a positive test does not distinguish between current illness and past exposure.
• Brucella canis:
• Brucella canis:
◦ Detection: Rapid slide agglutination detects surface antigen antibodies.
◦ Detection: Rapid slide agglutination detects surface antigen antibodies.
◦ False Positives: A 2-mercaptoethanol (2-ME) step is often included to eliminate nonspecific IgM reactions.
◦ False Positives: A 2-mercaptoethanol (2-ME) step is often included to eliminate nonspecific IgM reactions.
◦ Confirmation: Positive results must be confirmed with PCR or blood culture.
◦ Confirmation: Positive results must be confirmed with PCR or blood culture.
V. Heartworm Testing
V. Heartworm Testing
• Dogs:
• Dogs:
◦ Mechanism: Detects female heartworm antigen; male-only infections will not be detected.
◦ Mechanism: Detects female heartworm antigen; male-only infections will not be detected.
◦ Timing: Antigen is detectable roughly 5 months after infection.
◦ Timing: Antigen is detectable roughly 5 months after infection.
◦ Post-Treatment: Antigenemia can persist for up to 6 months after adulticide therapy.
◦ Post-Treatment: Antigenemia can persist for up to 6 months after adulticide therapy.
• Cats:
• Cats:
◦ Challenge: Cats have lower worm burdens, often only one or two worms, and frequently have single-sex infections.
◦ Challenge: Cats have lower worm burdens, often only one or two worms, and frequently have single-sex infections.
◦ Protocol: Antigen tests are less sensitive (50%–80%); therefore, combination testing for both antigen and antibody is recommended for the highest accuracy.
◦ Protocol: Antigen tests are less sensitive (50%–80%); therefore, combination testing for both antigen and antibody is recommended for the highest accuracy.
Reproductive and Endocrine Testing
Reproductive and Endocrine Testing
• Pregnancy (Relaxin):
• Pregnancy (Relaxin):
◦ Specific Hormone: Relaxin is produced by the placenta and is detectable around day 20–25 post-fertilization.
◦ Specific Hormone: Relaxin is produced by the placenta and is detectable around day 20–25 post-fertilization.
◦ Accuracy: High specificity means it is not found in nonpregnant or pseudopregnant animals, though false negatives can occur with very small litters.
◦ Accuracy: High specificity means it is not found in nonpregnant or pseudopregnant animals, though false negatives can occur with very small litters.
• Ovulation Timing (LH & Progesterone):
• Ovulation Timing (LH & Progesterone):
◦ LH Surge: Occurs 2 days before ovulation; testing must be daily to catch the brief peak.
◦ LH Surge: Occurs 2 days before ovulation; testing must be daily to catch the brief peak.
◦ Progesterone: Increases modestly before ovulation and more significantly on the day of ovulation (4–10 ng/mL); testing is done every 2–3 days.
◦ Progesterone: Increases modestly before ovulation and more significantly on the day of ovulation (4–10 ng/mL); testing is done every 2–3 days.
◦ Ovariectomy Status: Low LH indicates an intact (nonovariectomized) animal, while high LH (>1 ng/mL) is seen in both ovariectomized animals and those about to ovulate.
◦ Ovariectomy Status: Low LH indicates an intact (nonovariectomized) animal, while high LH (>1 ng/mL) is seen in both ovariectomized animals and those about to ovulate.
• Thyroxine (T4):
• Thyroxine (T4):
◦ Use: Screening for canine hypothyroidism or feline hyperthyroidism.
◦ Use: Screening for canine hypothyroidism or feline hyperthyroidism.
◦ Testing Nuance: While in-house semiquantitative ELISA exists, total T4 is 99% protein-bound; Free T4 levels are considered more accurate for thyroid function.
◦ Testing Nuance: While in-house semiquantitative ELISA exists, total T4 is 99% protein-bound; Free T4 levels are considered more accurate for thyroid function.
Neonatal Passive Transfer
Neonatal Passive Transfer
• Foal Immunoglobulin (IgG):
• Foal Immunoglobulin (IgG):
◦ Optimal Levels: >800 mg/dL is optimal, while <200 mg/dL indicates failure of passive transfer.
◦ Optimal Levels: >800 mg/dL is optimal, while <200 mg/dL indicates failure of passive transfer.
◦ Rapid Tests: Include zinc sulfate turbidity, glutaraldehyde clot, and ELISA.
◦ Rapid Tests: Include zinc sulfate turbidity, glutaraldehyde clot, and ELISA.
• Calf Immunoglobulin:
• Calf Immunoglobulin:
◦ Optimal Levels: Adequacy is defined as >1,000 mg/dL.
◦ Optimal Levels: Adequacy is defined as >1,000 mg/dL.
◦ Methods: Refractometry (measuring total protein, where 5.2 g/dL is roughly 1,000 mg/dL IgG) is a reliable indicator in well-hydrated calves.
◦ Methods: Refractometry (measuring total protein, where 5.2 g/dL is roughly 1,000 mg/dL IgG) is a reliable indicator in well-hydrated calves.
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