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Operating Instructions

Neospora-p38 ELISA Cattle Enzyme immunoassay for the detection of IgG antibodies to Neospora caninum




The Neospora-p38 ELISA Cattle is an enzyme immunoassay (ELISA) in microtitre plate format. It detects IgG antibodies to against Neospora caninum in serum samples of cattle. Neospora caninum is a cyst-forming coccidian parasite recently identified as a cause of abortion in cattle.


The Neospora-p38 ELISA Cattle has an extremely high sensitivity and specificity, compared to other Neospora-ELISA tests. This is based on the use of a single highly purified antigen protein p38, named after the molecular weight of 38 kilo Dalton (Schares et al. 2000).

Neosporosis caused by Neospora caninum has been recognized as a protozoan disease since 1988. The parasite undergoes a life cycle consisting of various stages, among them: sporozoites, tachyzoites and bradyzoites (enclosed in tissue cysts). During pregnancy bradyzoites can be activated and convert to tachyzoites which efficiently crosses the placenta and infect foetuses. The clinical findings are abortion, birth of calves with neurological signs such as weakness and ataxia, or birth of a symptom free but chronically infected calf (Conraths & Schares, 1999). During the 1990ies neosporosis has been recognised as an important reason of stillbirths and abortions in cattle in USA. Studies from California have indicated that 20-45% of all bovine abortions were attributable to neosporosis; studies from Europe indicate 11.9-12.5% (De Meeschmann et al., 2000; Davison et al, 1999).


The microtitre plate is coated with a Neospora p38 antigen.

During serum incubation specific anti-Neospora antibodies are bound to the immobilised antigen. Unbound material is removed by washing. The added enzyme-conjugate binds to the antigen-IgG antibodies complex. Unbound enzyme-conjugate is removed again by washing. Newly formed immune complexes are incubated with TMB substrate solution leading to the development of a blue colour. Blue colour turns into yellow by stopping the enzymatic reaction with stopping solution. The colour reaction correlates with the amount of Neospora antibodies in the serum sample. The diagnostic assessment is made by the comparison of the extinction values of tests and controls.


  • Microtitre plate (1 plate): Coated with Neospora p38 antigen (inactive), 12 stripes with 8 wells each = 96 wells per plate (single wells can be detached and used).
  • Sample diluent (1 bottle, 50 ml): Buffer, preserved with sodium azide, ready-to-use.
  • Washing solution concentrate (1 bottle, 50 ml): Phosphate buffered, 10-fold concentrated, preserved with ProClin 300.
  • Positive control serum PC (1 bottle, 1.5 ml): Serum of Neospora caninum -infected cattle, preserved with sodium azide, ready-to-use.
  • Negative control serum NC (1 bottle, 1.5 ml): Serum of not Neospora caninum -infected cattle, preserved with sodium azide, ready-to-use.
  • Conjugate solution (1 bottle, 12 ml): Horseradish peroxidase-conjugated anti-bovine IgG immunglobulins, ready-to-use, preserved with ProClin 300.
  • Substrate solution (1 bottle, 12 ml): TMB solution, (TMB = 3,3‘,5,5‘-tetramethylbenzidine), ready-to-use, preserved with kathon.
  • Stopping solution (1 bottle, 12 ml): Sulphuric acid, 1 mol/l, ready-to-use, Attention: Corrosive !


ProClin 300, kathon and sodium azide are toxic substances. Do not swallow, avoid contact with skin and mucous membranes.

Sulphuric acid irritates eyes and skin. Upon contact with eyes, rinse thoroughly with water and consult a physician!



Beakers, pipettes, pipette tips, reagent reservoirs, redistilled water, photometer for microtitre plates with a 450 nm filter


1. Use of microtitre plates

The microtitre plate is delivered in the foil bag with re-closure. In addition, there is a dry bag in the packaging with indicator. It should be stored at 4°C - 7°C all time

The microtitre plate consists of 12 stripes with 8 wells each. The number of wells to be used is equal to the number of samples to be measured, plus two wells for the control measurements. The wells have to be taken to room temperature (18 to 25° degrees Celsius) before use.

It is absolutely crucial to close again the foil bags after every usage. A colour change of the dry bag content of blue to red indicates too high relative air humidity in the foil bag. High humidity in the bag can affect the quality of the microtitre plate coated with antigen. The functionality of the bag should be checked and if necessary the dry bag should be replaced.


2. Preparation of test reagents

Before use bring all reagents to room temperature (18 – 25°C) and mix thoroughly. Return unused reagents to the refrigerator (store at 4°C – 7°C). Take only required amount of conjugate solution out of the vial and bring it to room temperature. Remaining conjugate should be continuously stored at 4?C – 7°C.

Dilute washing solution concentrate with distilled water 1 : 10 (1 part washing solution concentrate + 9 parts water). Diluted washing solution is ready to use and can be stored at 4º – 7ºC up to one week and at –20ºC up to three months.


3. Sample preparation

Suitable for the test is fresh, cool stored blood serum or frozen and defrosted blood serum. Thawing of blood serum should be rapidly carried out at room temperature or at 37°C. All sera, especially the thawed sera, should be mixed thoroughly before use.

Give 5 µl of blood serum in 495 µl of sample diluent (1:100).
Mix the dilution thoroughly.


4. Test procedure

  • All reagents should be brought to room temperature (18 to 25°C) before use.
  • Remove the required number of wells out of the foil pouch and place in the provided frame. Reseal pouch tightly to exclude moisture and return it to the fridge immediately. Before use bring the required wells to room temperature.
  • Add 100 µl of control sera (positive control serum PC and negative control serum NC) into the wells (single test run).
  • Add 100 µl of each diluted serum samples into a well (single test run).
  • Cover the plate and incubate at room temperature for 60 minutes.
  • Pour out or aspirate contents of the wells. Wash thoroughly by filling each well with 300 µl diluted washing buffer. Pour out or aspirate all fluid from wells after each wash. Repeat four times more (five washing steps in total).
  • Add 100 µl of conjugate solution into each well.
  • Cover the plate and incubate at room temperature for 60 minutes.
  • Pour out or aspirate contents of the wells. Wash thoroughly by filling each well with 300 µl diluted washing buffer. Pour out or aspirate all liquid from wells after each wash. Repeat four times more; five washing steps in total.
  • Add 100 µl of substrate solution into each well.
  • Cover the plate and incubate at room temperature for exactly 15 minutes. Clock the time after filling the first well.
  • Add 100 µl of stopping solution into each well. Do this in the same succession in which you have added the substrate solution.
  • Shake thoroughly. Measure the extinction values (OD) with a photometer at 450 nm within 10 min after adding of the stop solution.


1. Test validity

  • Calculate the percentage (P) of optical density of negative control serum (ODNC) from the following formula:

P = (ODNC x 100) / (ODPC)

(ODPC = optical density of positive control serum PC)

  • The test procedure is valid if the following is fulfilled:

ODPC > 0.5 < 2.8

P < 20


2. Calculation of test results

  • Subtract the optical density of negative control serum NC (ODNC) from the optical density of positive control serum PC (ODPC) as well as from the optical density of tested serum samples (ODsample).

ODPC, corr = ODPC – ODNC

ODsample, corr = ODsample – ODNC

  • Calculate the percentage of optical density of samples ( = test results, TR) from the following formula:

TR = (OPsample, corr x 100) / (ODPC, corr)


3. Interpretation of test results

TR < 8 = negative
TR 8 - 13 = unequivocal
TR > 13 = positive


Store all reagents at 4°C - 7°C and bring them to room temperature (18°C - 25 °C) just before usage. Do not expose substrate solution to direct sunlight. The test components should not be contaminated or mixed with reagents from other lots. Do not use any components of the test kit after expiry date.

Only persons with laboratory experience should perform the ELISA test. Adequate diligence should be taken in handling ELISA tests. For correct performance and results clean materials, precise pipetting, washing and keeping exact incubation times are necessary.

Some of the components contain toxic substances (sodium azide, thiomersal, ProClin300). At disposal of waste material the corresponding laws and regulations have to be taken into account.

The test kit has been produced for veterinary and in-vitro diagnostic use only.