Diagnostic kit for stallion infection using the method of complement bond reaction (CBR)

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Composition:  

The kit contains:

Trypanosoma equiperdum antigen - lyophilised mass of disintegrated trypanosomes.

The antigen is calibrated using a standard positive and negative serum, its capacity being adjusted so that in working dilution (4 x more concentrated than its titre) it provides a visible reaction with positive serum containing a minimum amount of specific antibodies in the dilution of 1:4. In working dilution it should not be anti-complementary and show false positive reactions with negative serum.

Control positive serum:

• Rabbit serum containing specific complement-fixing antibodies against Trypanosoma equiperdum preserved with phenol. It is calibrated using a standard antigen, its capacity being adjusted so that it exhibits a positive reaction in the dilution 1:16 at a minimum.

Haemolytic amboceptor:

• Rabbit serum containing thermostable antibodies against sheep blood cells, preserved with phenol.

Guinea pig complement:

• Guinea pig serum.

Solution for dilution:

• Solution to re-hydrate the lyophilised antigen and complement to the original volume.
• Concentrated Mayer’s buffer saline solution.

Indication:

For serological diagnostics of stallion infection using CBR.

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Performing the test:

Before use, antigen and guinea pig complement are diluted to the original volume (1 ml) using a dilution solution that is a part of the kit. For all other dilutions performed during the test the Mayer’s concentrated solution is to be diluted with sterile re-distilled water in a ratio of 1+4 before use.

Haemolytic amboceptor and complement should be titrated before titration itself.

Titration of haemolytic amboceptor:

Prepare 1:1000 basic dilution and from this an auxiliary dilution series of 1:10,000 or more. Starting with the highest dilution, pipette 0.2 ml of amboceptor of appropriate dilution in test tubes of each of the titration series. Pipette 0.2 ml of 2% suspension of ram blood cells, 0.4 ml of Mayer’s solution, and 0.2 ml of complement diluted 1:20 in all test tubes. At the same time complete the series with three test tubes of common controls: amboceptor, complement, and blood cells. Shake all tubes thoroughly and place immediately in water bath at 37ºC. Read the results after a 30-minute incubation.

Auxiliary dilution of amboceptor         

Test tube No.:	1	2	3	4	5	6	7	8	9	10
Final dilution of amboceptor	1000	2000	3000	4000	5000	6000	7000	8000	9000	10000
Amboceptor 1:1000 in ml	1	0.5	0.5	0.5	0.5	0.25	0.25	0.25	0.25	0.25
Mayer’s solution ml	-	0.5	1.0	1.5	2.0	1.25	1.5	1.75	2.0	2.25

 

Dilution of amboceptor	1000	2000	3000	4000	5000	6000	7000	8000	9000	10000	K1	K2	K3
Doses of individ. dilution in ml	0.2	0.2	0.2	0.2	0.2	0.2	0.2	0.2	0.2	0.2	0.2	-	-
											1 : 100
Mayer’s solution in ml	0.4	0.4	0.4	0.4	0.4	0.4	0.4	0.4	0.4	0.4	0.6	0.6	0.8
Guinea pig complement 1:20 in ml	0.2	0.2	0.2	0.2	0.2	0.2	0.2	0.2	0.2	0.2	-	0.2	-
2% suspension of sheep blood cells in ml	0.2	0.2	0.2	0.2	0.2	0.2	0.2	0.2	0.2	0.2	0.2	0.2	0.2
20-minute incubation in water bath at the temperature of 37 ºC.

• K 1 – amboceptor control
• K 2 – complement control
• K 3 – blood cell control

Evaluation of reaction:

• ++++                - complete inhibition of haemolysis /+4/
• +++, ++, +        - inhibition of haemolysis in gradually decreasing intensity /+3, +2, +1/
• -                      - 100% haemolysis – complete lysis of blood cells

A minimum haemolytic dose of amboceptor (MHD) is contained in 0.2 ml of the highest dilution of amboceptor that in the abundance of complement totally dissolves 0.2 ml of the suspension of ram blood cells (the last totally clear test tube). To sensibilise the blood cells, amboceptor is diluted so that it contains 4 MHD in  0.2  ml. The haemolytic system is prepared by mixing 2% suspension of blood cells with the same volume of amboceptor in the working dose of 4 MHD.

Complement titration:

It is diluted immediately before use using chilled dilution solution and is stored in the fridge. The basic dilution to proceed from is 1:25. Complement titration is done according to the table.

Tube No.	1	2	3	4	5	6	7	8	9	10	K1	K2	K3
Complement1 + 24	0.20	0.18	0.16	0.14	0.12	0.10	0.08	0.06	0.04	0.02	0.20	-	-
Mayer’s solution	0.40	0.42	0.44	0.46	0.48	0.50	0.52	0.54	0.56	0.58	0.60	0.60	0.80
Haem. amboceptor 4 MHD	0.20	0.20	0.20	0.20	0.20	0.20	0.20	0.20	0.20	0.20	-	0.20	-
2% ram blood cells	0.20	0.20	0.20	0.20	0.20	0.20	0.20	0.20	0.20	0.20	0.20	0.20	0.20
Incubation in water bath for 30 minutes at 37 ºC
% complem.	4%	3.6	3.2	2.8	2.4	2.0	1.6	1.2	0.8	0.4	++++	++++	++++

Results should be read after 30-minute incubation in a water bath at 37ºC. Titre of complement is the smallest amount of complement that, under given conditions, is able to dissolve all blood cells of the haemolytic system. Complement in the dose of 1.5 MHD is used as working dilution (e.g. complement titre is 2.4%, working dilution is 3.6%).

Used controls

• K 1 – complement control
• K 2 – amboceptor control
• K 3 – blood cell control

Examined serum should be inactivated before titration in a water bath at 60oC for the period of 30 minutes. Examined serum should be diluted for titration as follows: 1 + 3 (titre 1 : 4), 1 + 7 (titre 1 : 8), etc. The same amount of antigen in its working dilution should be added to prepared dilutions of serum followed by the addition of the same part of titrated complement (1.5 MHD), and the serum is then incubated in water bath at 37oC for the period of 30 min. Then a double amount of the haemolytic system is added (the same part of 2% ram blood cells and haemolytic amboceptor - 4 MHD). Incubation is performed in a water bath at 37°C for 30 min. The test for anti-complementarity of the examined serum is performed in the same way. In this series antigen is replaced with Mayer’s dilution solution. A part of titration is represented by control of the guinea pig complement, haemolytic system, and the used antigen with control positive serum in its working dilution. 

Performing the test: according to the table

Test serum	Dilution	1:4	1:8	1:16	1:32	1:64	1:128	1:256	1:512	K1	K2	K3	K4
	Dose	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	-	-	contser 0.1	-
Antigen Trypanosoma equiperdum in working dilution		0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	-	-	0.1	0.1
Guinea pig complement 1.5 MHD		0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1	-	0.1	0.1
Incubation in water bath at 37ºC for 30 minutes
Haemolytic system		0.2	0.2	0.2	0.2	0.2	0.2	0.2	0.2	0.2	0.2	0.2	0.2
Mayer’s solution										0.2	0.3	-	0.2
Incubation in water bath at 37ºC for 30 minutes											++++	++++	-

Evaluation of reaction: 

• ++++                  - total inhibition of haemolysis 
• +++, ++, +          - inhibition of haemolysis in gradually decreasing intensity
•  -                        - complete lysis of blood cells

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Package:

The kit contains:

• Antigen Trypanosoma equiperdum - lyophilised        – 1 ml
• Positive control serum                                            – 1 ml
• Haemolytic amboceptor                                          – 1 ml
• Guinea pig complement                                          – 1 ml
• Solution for dilution                                                – 2 ml
• Concentrated Mayer’s buffer saline solution.             - 100ml

Storage:

In a dry dark place at the temperature of 2 to 8 °C.

Disposal of the kit:The packages or any remainders of the preparation have to be disposed of pursuant to the regulations in force.

Expiry:

1 year

Note: Expiration of the kits is determined by the expiration date of the complement. Other components contained in the kit may be used within the expiration date stated on them. Guinea-pig complement and haemolytic amboceptor may also be purchased separately.