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SAM 405.04

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 405

Supplemental Assay Method for Detecting Lymphoid Leukosis 

Biocontamination by the COFAL Test

Date: November 28, 2014

Number: SAM 405.04

Supersedes: SAM 405.03, March 10, 2014

Standard Requirement: 9 CFR 113.31

Contact: Alethea M. Fry, (515) 337-7200

Claude A. Hovick

Approvals: /s/Geetha B. Srinivas Date: 26Jan15

Geetha B. Srinivas, Section Leader

Virology

/s/Byron E. Rippke Date: 29Jan15

Byron E. Rippke, Director

Policy, Evaluation, and Licensing

Center for Veterinary Biologics

/s/Rebecca L.W. Hyde Date: 29Jan15

Rebecca L.W. Hyde, Section Leader

Quality Management

Center for Veterinary Biologics

United States Department of Agriculture

Animal and Plant Health Inspection Service

P. O. Box 844

Ames, IA 50010

Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by USDA 

and does not imply its approval to the exclusion of other products that may be suitable.
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Table of Contents

1. Introduction

2. Preparation for the Test

2.1 Personnel qualifications/training

2.2 Preparation of equipment/instrumentation

3. Part I. Propagation of Extraneous Lymphoid Leukosis Virus in Chick 

Embryo Fibroblasts (CEFs)

3.1 Equipment/instrumentation

3.2 Supplies

3.3 Reagents

3.4 Primary CEF preparation

3.5 Secondary CEF preparation

3.6 Preparation and inoculation of test material

3.6.1 Avian encephalomyelitis (AE) vaccine (liquid glycerinated 

product)

3.6.2 AE vaccine, lyophilized 

3.6.3 Hemorrhagic enteritis virus and marble spleen disease vaccine

3.6.4 Infectious bronchitis vaccine, lyophilized or frozen

3.6.5 Infectious bursal disease (IBD) vaccine, lyophilized or frozen

3.6.6 Laryngotracheitis vaccine, lyophilized

3.6.7 Marek's disease (MD) vaccine, cell associated, frozen

3.6.8 MD vaccine, lyophilized

3.6.9 Newcastle disease vaccine and combination Newcastlebronchitis vaccine, lyophilized or frozen

3.6.10 Pox and AE-pox combination vaccine

3.6.11 Reovirus or tenosynovitis virus vaccine, lyophilized or frozen

3.6.12 Recombinant virus vaccine

3.7 First and second harvest

3.8 Final harvest

4. Part II. Detection of Extraneous Lymphoid Leukosis Virus Group Specific 

(gs) Antigen by a Microtiter Complement Fixation (CF) Test

4.1 Equipment/instrumentation

4.2 Reagents/supplies
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4.3 Preparation and standardization of reagents

4.3.1 Preparation of Veronal Buffered Diluent (VBD)

4.3.2 Preparation and standardization of 2.8% percent sheep red 

blood cell suspension

4.3.3 Preparation of hemoglobin color standards

4.3.4 Hemolysin titration

4.3.5 Preparation of sensitized cells

4.3.6 Complement (C') titration

4.3.7 Serum and antigen titrations

4.4 Detection of gs antigen in test material

5. Report of Test Results

6. Repeat Tests

7. References

8. Summary of Revisions

Appendices

I. Materials

II. Counting cells with a hemacytometer

III. Formulating cell suspensions
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1. Introduction

This Supplemental Assay Method (SAM) describes a procedure for the detection of lymphoid 

leukosis virus biocontamination in live virus poultry vaccines. The Complement Fixation for 

Avian Leukosis (COFAL) test is a 2-part procedure: Part I. Propagation of lymphoid leukosis 

virus, if present, in Chick Embryo Fibroblast (CEF) cell culture; and Part II. Detection of the 

group-specific (gs) antigen by a microtiter complement-fixation technique.

2. Preparation for the Test

2.1 Personnel qualifications/training

The personnel must have experience or training in this protocol. This includes knowledge 

of safe and aseptic biological laboratory techniques and preparation, proper handling and 

disposal of biological agents, reagents, tissue culture samples, and chemicals. The 

personnel must also have training in the operation of the necessary laboratory equipment 

required for this assay.

2.2 Preparation of equipment/instrumentation

Operate and monitor all equipment/instrumentation according to manufacturers’ 

instructions. Maintain aseptic conditions in a laminar flow biological safety cabinet, and 

use sterile instruments and wear sterile gloves when appropriate. 

3. Part I. Propagation of Extraneous Lymphoid Leukosis Virus in CEFs

3.1 Equipment/instrumentation

Equivalent equipment or instrumentation may be substituted for any brand name listed 

below.

3.1.1 Laminar Flow Biological Safety Cabinet (NuAire Inc., Labgard)

3.1.2 Humidified, rotating egg incubator (Midwest Incubators, Model 252)

3.1.3 Water-jacketed incubator with a humidified 5% CO2 atmosphere and 

temperature set at 37°C (Forma Scientific, Model No. 3158)

3.1.4 Roller apparatus (to rotate roller bottles)

3.1.5 Centrifuge (Beckman J6-MI, JS-4.2 rotor)
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3.1.6 Vacuum pump (Curtin Matheson Scientific, Inc.)

3.1.7 Bunsen burner (Hanau Engineering Co., Touch-O-Matic)

3.1.8 Scissors, sterile (Roboz RS-6800)

3.1.9 Curved tip forceps, sterile (V. Mueller SU 2315)

3.1.10 Magnetic stir bars (sterile) and a stir plate

3.1.11 Hemacytometer (American Optical)

3.2 Supplies

Equivalent supplies may be substituted for any brand name listed below. All supplies 

must be sterile.

3.2.1 Tissue culture dish, 150 x 10-mm (Falcon, Cat. No. 1058)

3.2.2 Tissue culture dish, 100 x 20-mm (Falcon, Cat. No. 3003)

3.2.3 Plastic funnel covered with 4 layers of fine gauze

3.2.4 Conical tube, polypropylene, 29 x 114-mm, 50-mL (Sarstedt, Cat. No. 

62.547.205)

3.2.5 Centrifuge tubes, polypropylene, 250-mL (Corning, Cat. No. 25350)

3.2.6 Roller bottles, 2000-mL, 850-cm

2

, with screw-caps (Becton Dickinson, 

Cat. No. 3007)

3.2.7 Trypan Blue

3.2.8 Snap-cap tubes, 12 x 75-mm (Falcon, Cat. No. 2058)

3.2.9 Cell lifters (Costar, Cat. No. 3008)

3.2.10 Filtering flask, 1-L, with hose and cannula

3.2.11 Serological pipettes

3.2.12 Erlenmeyer flasks
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3.2.13 Syringes and needles

3.2.14 Membrane filters, 0.22 μ (Millipore, Cat. No. SLGA025OS)

3.2.15 Membrane filters, 0.45 μ (Millipore, Cat. No. SLHA0250S)

3.2.16 Membrane filters, 5.0 μ (Millipore, Cat. No. SLAP02550)

3.3 Reagents

Equivalent reagents may be substituted. All reagents must be sterile.

3.3.1 Two dozen 9- to 11-day-old Specific-Pathogen-Free (SPF) chick embryos 

from a genetic line of chickens (C/O) which are susceptible to all lymphoid 

leukosis virus subgroups

3.3.2 Growth Medium

Medium 199 with Earles salts 1 L

Bacto tryptose phosphate broth 50 mL

NaHCO3 1.5 g

Penicillin (potassium G) 100,000 units

Streptomycin sulfate 100 mg

Fetal bovine serum (FBS) (inactivated) 30-55 mL

Fungizone (optional) 2 mg

Adjust pH to 7.3 with NaHCO3 solution

Note: Better cell growth and maintenance may occur if L-glutamine 

(10 mL/1000 mL growth medium = 1% L-glutamine) is added within 3 days 

prior to use of the medium.

If growth medium is used to wash embryonic tissues prior to trypsinization, 

omit the FBS and L-glutamine. 

3.3.3 Puck's Saline A

NaCl 8.0 g

KCl 0.4 g

Glucose 1.0 g

Phenol Red (0.5% solution) 1.0 mL

q.s. with distilled or deionized water (DW) 1 L

Adjust pH to 7.2 with NaHCO3 solution.
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3.3.4 Ethylenediamine tetraacetate (EDTA) stock solution (1.0%), also 

referred to as versene

Add 1.0 g ethylenedinitrilo tetraacetic acid disodium salt to 100 mL Puck's Saline 

A.

3.3.5 Trypsin solution (0.25%)

Add 2.5 g trypsin (1:250) to 1 L Puck's Saline A. 

Add 0.35 g NaHCO3 per L.

Adjust pH to 7.4 with NaHCO3 solution.

Note: For trypsinizing embryos, use 0.25% trypsin solution as is.

For trypsinizing primary cultures, add 2.0 mL of 1% EDTA to 100 mL

0.25% trypsin solution, or 0.25% trypsin may be used as is.

For trypsinizing secondary or higher passage chick embryo cell cultures, mix 

0.25% trypsin 1:5 (1 part trypsin plus 4 parts Puck’s Saline A) in Puck's 

Saline A. Then add 2.0 mL of 1.0%. EDTA solution per 100 mL trypsin 

solution. This makes a solution containing 0.05% trypsin and 0.02% EDTA. 

Add a few drops NaHCO3 solution to bring the pH up to 7.2-7.4.

3.3.6 70% ethanol

3.4 Primary CEF preparation

Primary CEF cultures can be prepared from 9- to 11-day-old genetically susceptible 

(C/O) embryos from a COFAL-negative chicken flock in the following manner, or 

alternate methods of preparation are acceptable:

3.4.1 Disinfect the air cell end of the egg with 70% ethanol, flame, and break 

open the shell with sterile forceps. Use the forceps to open the membranes, lift out 

the embryo, and place it in a sterile disposable 150 x 10-mm tissue culture or petri 

dish. Four to 24 embryos may be prepared together. Remove (and discard) the 

heads and viscera of the embryos with sterile scissors and forceps. Wash the 

embryos several times with a 0.25% trypsin solution (room temp), a physiological 

saline solution, or growth medium without FBS or L-glutamine. Place the washed 

embryos in a sterile dry 150 x 10-mm tissue culture or petri dish and mince them 

thoroughly using sharp sterile scissors.

3.4.2 To further wash, put the minced tissue into a trypsinizing flask containing 

50 mL growth medium without FBS or L glutamine and a magnetic stir bar. Place 
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the flask on a stir plate and stir with a moderate vortex for 5 minutes. Allow the 

cells to settle and decant (and discard) the supernatant and repeat.

3.4.3 To trypsinize the tissues, first rinse the residual media from the cells by 

adding 25 mL of the 0.25% trypsin solution, and then immediately decant the 

trypsin solution. Next, add 50 mL of the 0.25% trypsin solution to the flask and 

mix on a magnetic stir plate for 20 minutes. (Set stir plate to produce a moderate 

vortex.)

3.4.4 Place a sterile gauze wrapped funnel into the opening of a 250-mL conical 

centrifuge tube. To stop the trypsinizing action on the cells, pour 2 mL of FBS 

through the gauze and then pour the trypsinized contents of the flask through the 

gauze funnel into the centrifuge tube. Bring the total volume to approximately 

125 mL with growth media. Centrifuge for 10 minutes at 250 x g with the 

temperature set at 10°C. 

3.4.5 Observe and record the volume of packed cells, and then pour off the 

supernatant. Dilute the cells approximately 1:300 with growth medium. Plant 

200 mL of diluted cell suspension in each 1000-mL roller bottle. Tighten the cap 

and incubate for 4 days in an incubator set at 37°C. (Alternately, if tissue culture 

flasks are planted, adjust the volume of cell suspension appropriately, leave the 

caps loose, and incubate in a humidified incubator with the temperature set at 

37°C and the atmosphere set at 5% CO2 for 4 days.) After 4 days, the cell sheet 

should be well proliferated and ready to split.

3.5 Secondary CEF preparation

Secondary CEF cultures can be prepared in the following manner, or alternate methods of 

preparation are acceptable:

3.5.1 Decant the medium from 1 roller bottle of primary CEFs, pipetting any 

remaining media with a 5-mL pipette, and add 15 mL of the 0.25% trypsin 

solution prewarmed to 37°C. Rotate the bottle until the cells begin to detach, 

approximately 1 minute. (The proper length of time will be learned by experience. 

Too short a time will result in large clumps of cells in the new suspension.) 

Decant the trypsin and, with an open hand, strike the side of the roller bottle until 

most of the cells become detached. Rinse the inside of the roller bottle with 15 

mL of growth medium and swirl. Pipette the suspension with the cells into an 

empty Erlenmeyer flask. Repeat twice more for a total of 3 rinses. Repeat the 

above steps separately for each additional roller bottle.

3.5.2 Pour the combined cell suspension from the Erlenmeyer flask through a 

funnel covered with 4 layers of fine gauze into a second Erlenmeyer flask 

containing a sterile stir bar and 25 mL of growth medium for each roller bottle 

used. Then rinse the funnel with an additional 25 mL of medium for each roller 

bottle used. Thoroughly mix the cell suspension.
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3.5.3 Make a cell count with a hemocytometer. (A total cell count using crystal 

violet stain is satisfactory.) See the Appendices. Adjust the volume so that the 

cell concentration is approximately 500,000 cells per mL.

3.5.4 Plant the secondary cell suspension into the vessels to be used for the test 

(usually 60-mm or 100-mm plastic tissue culture dishes). Use 5 mL of the 

suspension for a 60-mm dish and 15 mL of the suspension for a 100-mm dish. 

The cultures are now ready to be inoculated with test materials.

3.6 Preparation and inoculation of test material

This protocol is designed primarily to detect infective lymphoid leukosis virus 

contamination in live virus poultry vaccines. Preparation procedures described are those 

which are least complicated, but still have been found in most cases to be adequate for 

the purpose. These procedures are tentative and will of necessity be changed as more 

knowledge and experience dictates. A suggested procedure for each type of vaccine is 

described separately. If the test materials are to be diluted for inoculation, regular growth 

medium or medium without serum can be used. In each case, add the inoculum to the 

plates in addition to the growth medium already in the plates. The test material may be 

inoculated immediately after planting the cell cultures if experience has shown the 

material used will not be toxic to chick embryo fibroblasts. After inoculation, incubate 

the cultures in a humidified incubator set at 37°C and containing 5% CO2. Sixteen to 24 

hours later, remove the fluids and replace with fresh growth medium added at the original 

planting volume of 5 mL per 60-mm plate or 15 mL per 100-mm plate.

Note: With materials that may be somewhat toxic, allow the cell sheets to attach and 

grow for 18 hours prior to adding the test inoculum, and then leave inoculum on the 

cells for 4 to 24 hours depending on the toxic effect.

3.6.1 Avian encephalomyelitis (AE) vaccine (liquid glycerinated product)

This product tends to be somewhat toxic to the cell cultures, and the procedure for 

inoculation needs to be adjusted accordingly. Dilute the vaccine with tissue 

culture medium so that 1000 doses are present in 20 mL total volume. Centrifuge 

the mixture for 30 minutes at 1800 x g.

The solids will be primarily in the bottom, the fat- containing portion on top, and 

an aqueous portion in the middle. Insert a pipette into the aqueous portion and 

remove more than 10 mL. Filter this through a 5.0-micron membrane filter. 
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Divide a quantity of filtrate equal to 500 doses (10 mL) among 5, 60-mm plates or 

2, 100-mm plates. With this product, it is not known how much, if any, the 

vaccine virus or other vaccine components interfere with infection of the cells by 

leukosis virus.

3.6.2 AE vaccine, lyophilized

Restore this product with 1.0 mL tissue culture medium per 100 doses vaccine. If 

the product contains coarse tissue elements, centrifuge the restored material for 20 

minutes at 1000 x g and filter through a 0.45-micron membrane filter. Divide an 

amount of inoculum equal to 500 doses among 5, 60-mm plates or 2, 100-mm 

plates. To date, none of the products of this type have adversely affected the cell 

cultures. How much, if any, interference to infection of the cells by leukosis virus 

is caused by this vaccine virus is not known.

3.6.3 Hemorrhagic enteritis virus and marble spleen disease vaccine

Thaw or restore and dilute these products with 1.0 mL tissue culture medium per 

100 doses vaccine. If the product contains coarse tissue elements, centrifuge the 

restored material for 20 minutes at 1000 x g and filter through a 0.45 micron 

membrane filter. Divide an amount of inoculum equal to 500 doses among 5, 60-

mm plates or 2, 100-mm plates. To date, none of the products of this type have 

adversely affected the cell cultures. How much, if any, interference to infection of 

the cells by leukosis virus is caused by these vaccine viruses is not known.

3.6.4 Infectious bronchitis vaccine, lyophilized or frozen

Thaw or restore and dilute these products with 1.0 mL tissue culture medium per 

100 doses vaccine. If the product contains coarse tissue elements, centrifuge the 

restored material for 20 minutes at 1000 x g and filter through a 0.45-micron 

membrane filter. Divide an amount of inoculum equal to 500 doses among 5, 60-

mm plates or 2, 100-mm plates. To date, none of the products of this type have 

adversely affected the cell cultures. How much, if any, interference to infection of 

the cells by leukosis virus is caused by this vaccine virus is not known.

3.6.5 Infectious bursal disease (IBD) vaccine, lyophilized or frozen

Thaw or restore and dilute this product with 1.0 mL tissue culture medium per 

100 doses vaccine. Centrifuge the restored material for 20 minutes at 1000 x g

and filter through a 0.45-micron membrane filter. Neutralize the IBD virus with 

high titer specific antiserum (antiserum must be free of leukosis-sarcoma viruses 

and their antibodies). Mix 2 mL of the restored vaccine (200 doses) with 3 mL of 

the antiserum and incubate at room temperature for 1 hour. In some instances, a 

higher proportion of antiserum may be necessary. After the incubation period, 

inoculate the mixture, equivalent to 200 doses, into the cell cultures, dividing the 

entire amount equally among 5, 60-mm plates or 2, 100-mm plates.
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3.6.6 Laryngotracheitis vaccine, lyophilized 

Restore this product with 1.0 mL tissue culture medium per 100 doses vaccine. 

Centrifuge the restored material for 20 minutes at 1000 x g, and then filter 

through a 0.22-micron membrane filter. Divide an amount of inoculum equal to 

500 doses among 5, 60-mm plates or 2, 100-mm plates. 

3.6.7 Marek's disease (MD) vaccine, cell associated, frozen

Thaw and dilute vaccine to 100 doses per 1.0 mL in tissue culture medium. 

Centrifuge 10.0 mL of the inoculum for 20 minutes at 1000 x g and filter through 

a 0.22-micron membrane filter. Incubate at 25°- 30°C for 20 to 30 minutes. 

Divide a quantity of filtrate equal to 500 doses among 5, 60-mm plates or 2, 100-

mm plates.

3.6.8 MD vaccine, lyophilized 

Restore this product with 1.0 mL tissue culture medium per 100 doses vaccine. 

Centrifuge the restored material for 20 minutes at 1000 x g and filter through a 

0.22-micron membrane filter. Neutralize 500 doses of the Marek's disease vaccine 

virus with high titer specific antiserum (antiserum must be free of leukosissarcoma viruses and their antibodies) and incubate at room temperature for 1 

hour. After the incubation period, inoculate the mixture into the cell cultures, 

dividing the entire amount equally among 5, 60-mm plates or 2, 100-mm plates.

3.6.9 Newcastle disease vaccine and combination Newcastle-bronchitis 

vaccine, lyophilized or frozen

Thaw or restore and dilute these products with 1.0 mL tissue culture medium per 

100 doses vaccine. Neutralize the Newcastle disease virus (NDV) with high titer 

specific antiserum (antiserum must be free of leukosis-sarcoma viruses and their 

antibodies). Mix 2 mL of the restored vaccine (200 doses) with 2 mL of the NDV 

antiserum and incubate at room temperature for 1 hour. If the Newcastle vaccine 

is of particularly high titer, a higher proportion of antiserum may be necessary. 

After the incubation period, inoculate the mixture into the cell cultures, dividing 

the entire amount equally among 5, 60-mm plates or 2, 100-mm plates.

3.6.10 Pox and AE-pox combination vaccine

An optimum method for effectively neutralizing, inactivating, or separating out 

pox virus has not been determined, but the following method may be attempted. 

Thaw or restore and dilute these products with 10.0 mL tissue culture medium per 

500 doses vaccine. Centrifuge for 20 minutes at 1000 x g and filter through a 

0.22-micron membrane filter. Divide an amount of inoculum equal to 500 doses 

among 5, 60-mm plates or 2, 100-mm plates.
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3.6.11 Reovirus or tenosynovitis virus vaccine, lyophilized or frozen

An optimum method for effectively neutralizing, inactivating, or separating out 

this virus has not been determined, but the following method may be attempted. 

Thaw or restore and dilute these products with 1.0 mL tissue culture medium per 

100 doses vaccine. Centrifuge the restored material for 20 minutes at 1000 x g

and filter through a 0.45-micron membrane filter. Neutralize the reovirus or 

tenosynovitis virus with high titer specific antiserum (antiserum must be free of 

leukosis-sarcoma viruses and their antibodies). Mix 2 mL of the restored vaccine 

(200 doses) with 2 mL of the antiserum and incubate at room temperature for 1 

hour. In some instances, a higher proportion of antiserum may be necessary. After 

the incubation period, inoculate the mixture, equivalent to 200 doses, into the cell 

cultures, dividing the entire amount equally among 5, 60-mm plates or 2, 100-mm 

plates).

3.6.12 Recombinant virus vaccine

Follow the same method as for the vectoring agent.

Note: With each test series, maintain both positive and negative controls. 

Positive controls consist of 2 sets of plates, 1 inoculated with a subgroup A 

lymphoid leukosis virus, and 1 inoculated with a subgroup B lymphoid 

leukosis virus. Maintain a set of uninoculated plates as a negative control.

In all cases, remove the inoculum and medium in 24 hours or less after 

inoculating test material and add fresh growth medium.

Allow the cultures to grow for another 4 or 5 days without further treatment 

unless the pH of the medium becomes acid, necessitating a medium change.

At the end of this time, the first harvest of test material is made and the 

remaining cells are subcultured.

3.7 First and second harvest

3.7.1 Using an inverted microscope, examine each dish for contamination by 

bacteria or molds and to observe the condition of the cell sheet. Discard all 

contaminated plates and those with poor cellular growth. Harvest samples and 

passage cells as per Sections 3.7.2-3.7.3. Alternate methods are acceptable 

provided appropriate samples are harvested and cells are split and transferred. It is 

not acceptable to seed the split cells onto a new cell sheet of CEFs.

Note: Process only 1 set of plates at a time and in the following order: 

Negative controls, test samples, and then positive controls.
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3.7.2 Tip the culture dishes slightly and, using a suction apparatus, remove all 

but approximately 0.25 mL of culture fluid from a 60-mm dish or 0.75 mL from a 

100-mm dish. Use a sterile cell lifter and scrape off 1/2 of the cell sheet from each 

plate into the remaining fluids. Remove and pool these cell suspensions for each 

set of test plates. Freeze these pools at -60°C or colder until the complement 

fixation (CF) test is conducted. 

3.7.3 Subculture the remaining cells in the following manner: Add 0.05% 

trypsin (trypsin/Puck's Saline, 1:5) plus 0.02% versene solution (3 mL per 60-mm 

dish, 5 mL per 100-mm dish) to each dish. Allow the trypsin solution to remain in 

contact with the cell sheet for 1 to 2 minutes, swirling occasionally, and then 

remove it immediately when the cells begin to detach from the plate. Let the 

dishes stand at room temperature until the cells loosen further. (Strike the plates 

sharply with the palm of the hand to facilitate cell detachment.) Add fresh growth 

medium to the dishes, and disperse the cells with gentle pipetting. Pool these cell 

suspensions, mix thoroughly, and plant into new dishes. Plant 1 new culture dish 

for each original culture dish (a 2-for-1 split as 1/2 the cells were saved for 

testing). Incubate these cultures at 37°C as before.

Note: The cultures will usually grow for 1 week without a medium change, 

but if the pH becomes too acid, the medium will have to be changed in the 

interim. At the end of this week period, handle the cultures exactly the same 

way as at the first subculture. Should the growth rate be so rapid that the cell 

sheet begins to peel before the scheduled time for subculture, an additional 

harvest and subculture may be made.

3.8 Final harvest

After this second subculture (postinoculation), maintain the cultures under the same 

conditions as before until the 21st day post inoculation, at which time a final harvest is 

conducted. At this final harvest, leave twice the fluid in each plate as before and scrape 

off the entire sheet with a cell lifter. Divide the final harvest (each test) and store a 

portion at -60°C or lower until after CF testing of the other portion is completed. If the 

CF testing of any particular series is inconclusive, the stored portion of the final harvest 

may be used as an inoculum for a new test series.

Cell culture fluids from each subculture or medium change are tested for extraneous 

lymphoid leukosis by the CF test. 
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4. Part II: Detection of Extraneous Lymphoid Leukosis Virus Group Specific (gs) 

Antigen by a Microtiter Complement Fixation (CF) Test

The CF test method described has been adapted from the Laboratory Branch Complement 

Fixation (LBCF) Method developed at the National Communicable Disease Center, Atlanta, 

Georgia. The LBCF method is described in detail in Public Health Monograph No. 74. A 

supplement to this is entitled, "A Guide to the Performance of the Standardized Diagnostic 

Complement Fixation Method and Adaptation to Micro Test." First edition, July 1, 1969, has 

been prepared by the same Laboratory Section. These documents may be available from Public 

Health Inquiries Branch, U. S. Public Health Service, Washington, D.C. 20201.

4.1 Equipment/instrumentation

Equivalent equipment or instrumentation may be substituted for any brand name listed 

below.

4.1.1 Centrifuge (Beckman J6-MI, JS-3.0 rotor)

4.1.2 Incubator CO2 (Forma Scientific, Model No. 3158)

4.1.3 Freezer, -70°C (Revco Scientific, Model No. ULT1790-7-ABA)

4.1.4 Water bath, 37°± 1°C

4.1.5 Mini-Orbital shaker (Bellco Glass, Cat. No. 7744-S0010)

4.1.6 Vortex mixer (Thermolyne Maxi Mix II, Model No. M37615)

4.1.7 Reading mirror (Cooke Engineering Co. Microtiter)

4.1.8 12-channel pipette (Labsystems Model Finnpipette Digital Multichannel 

50-300 μL)

4.1.9 Pipettes (Labsystems Model Finnpipette Digital 5-40 μL & 40-200 μL)

4.1.10 Plate cover roller

4.2 Reagents/supplies

Equivalent reagents or supplies may be substituted for any brand name listed below.

4.2.1 Guinea pig complement (Whittaker Bioproducts, Cat. No. 30-956J)

4.2.2 Anti-sheep hemolysin and diluent (Baltimore Biological Laboratory)
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4.2.3 Sheep blood (25 mL Alsevers + 25 mL sheep blood) 4 to 31 days after 

collection

4.2.4 DW

4.2.5 Graduated Kolmer centrifuge tube, 10-mL (Corning Cat. No. 8360 or 

Kimbal Cat. No. 45180)

4.2.6 Graduated cylinder, 100-mL

4.2.7 Erlenmeyer flasks, 125- and 250-mL

4.2.8 Beaker, 50-mL

4.2.9 Tissue culture 96-well U-shaped plate (Linbro, Cat. No. 76-013-05)

4.2.10 Adhesive plate cover (Dynatech Laboratories, Cat. No. 001-010-3501) 

4.2.11 Polypropylene conical tube, 50-mL, 29 x 114-mL, sterile, (Sarstedt, Cat. 

No. 62.547.205)

4.2.12 Disposable polystyrene centrifuge tubes, 15-mL, 17 x 100-mL tubes 

(Falcon, Cat. No. 2057) 

4.2.13 Disposable borosilicate serologic tubes, 3-mL, 12 x 75-mm

4.2.14 Snap-cap tubes, 3-mL, 12 x 75-mm

4.2.15 Disposable serological pipettes, 1-, 5-, 10- and 25-mL

4.2.16 Pipette tips

4.2.17 Log-log paper

4.2.18 Plain graph paper
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4.3 Preparation and standardization of reagents

4.3.1 Preparation of Veronal Buffered Diluent (VBD)

1. Preparation of the stock buffer solution (5X VBD)

a. Combine the following in a 2-L volumetric flask in the order 

listed:

NaCl 83.00 g

Na-5, 5-diethyl barbiturate 10.19 g

DW 1500 mL

1 N hydrochloric acid 34.58 mL

Stock solution containing 1.0 molar 

 MgC12 and 0.3 molar CaC12

 (20.3 gm MgCl2•6H2O and 

 4.4g CaC12•2H2O in 

 100 mL DW) 5.00 mL

b. Fill to the 2-L mark with DW. Mix thoroughly.

c. Check the pH of the stock buffer before refrigeration by making a 

1:5 dilution with DW (1 part 5X VBD plus 4 parts distilled water). The 

pH of the diluted stock (1X VBD) must be 7.3-7.4. If the pH is not in 

this range, discard and prepare fresh stock buffer.

2. Preparation of gelatin-water solution

a. Add 1.0 g of gelatin to 100 mL of DW. Bring to a boil to dissolve 

the gelatin.

b. Make up to 800 mL with sterile DW at room temperature.

c. Chill in the refrigerator. This solution should not be held longer 

than 1 week.

3. Preparation of VBD (lX) for daily use (containing 0.1% gelatin)

Add 4 volumes of gelatin water to 1 volume of 5X VBD stock buffer. 

Store in the refrigerator. VBD (1X) should not be stored longer than 

24 hours.

Note: The pH of the VBD (1X) must be 7.3 to 7.4.
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4.3.2 Preparation and standardization of 2.8% sheep red blood cell 

suspension

1. Washing sheep cells (collected and preserved in modified Alsever's 

solution, aged 4 days)

a. Add 2 or 3 volumes of cold VBD (1X) to each volume of 

preserved cells and centrifuge at 600 X g for 5 minutes. 

b. Carefully aspirate the supernatant fluid and the white cell layer 

without disturbing the erythrocytes.

c. Fill centrifuge tube again with cold VBD. Thoroughly resuspend 

the cells by gently mixing with a pipette. Centrifuge at 600 X g for 5 

minutes and repeat the process for a total of 3 washings. If the 

supernate is not colorless after the second washing, cells are too fragile 

and must not be used.

d. Resuspend the cells once more in cold VBD and centrifuge for 10 

minutes at 600 X g to pack the cells.

e. Record the volume of packed cells in the centrifuge tube, and 

aspirate the supernate. Care should be taken to remove as much fluid 

as possible without disturbing the cells.

2. Standardization of 2.8% cell suspension

a. Prepare the 2.8% cell suspension (approximately 670,000 

cells/mm3) by adding 34.7 volume of VBD to 1 volume of packed 

sheep cells (Illustration 1). Shake flask gently to insure even 

suspension of cells.
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ILLUSTRATION 1

Standardization of Sheep Red Blood Cells to 2.8%

Cell Pack Volume

(mL)

Adjust to

Total Volume with VBD (mL)

0.5 17.9

0.6 21.4

0.7 25.0

0.8 28.6

0.9 32.1

1.0 35.7

1.1 39.3

1.2 42.8

1.3 46.4

1.4 50.0

1.5 53.6

1.6 57.1

1.7 60.7

1.8 64.3

1.9 67.8

2.0 71.4

2.1 75.0

2.2 78.5

2.3 82.1

2.4 85.7

2.5 89.3

2.6 92.8

2.7 96.4

2.8 100.0

2.9 103.5

3.0 107.1

3.1 110.7

3.2 114.2

3.3 117.8

3.4 121.4

3.5 125.0

3.6 128.5

3.7 132.1

3.8 135.6

3.9 139.2

4.0 142.8

b. To check the density of the 2.8% cell suspension, pipette 

7.0 mL into a 10-mL graduated Kolmer centrifuge tube (either a 

Corning 8360 or Kimbal 45180 has acceptable tolerance) and 

centrifuge at 600 X g for 10 minutes. A 7.0 mL aliquot of properly 

prepared cell suspension should produce 0.2 mL of packed cells.
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c. When the volume of packed cells is under or above the 0.2 mL

point, the cell suspension should be adjusted. The quantity of VBD 

which must be added to or removed from the cell suspension is 

determined by the following formula, where PCV = actual packed cell 

volume of 7.0 mL

(PCV/0.20 mL) x 100 mL = corrected volume

Example 1, low density: PCV = 0.19 mL

0.19 mL x 100 mL = 95.0 mL

0.20 mL

Therefore, remove 5 mL of VBD from each 100 mL of cell 

suspension. (VBD may be removed by centrifuging an aliquot of the 

cell suspension and pipetting off the desired amount for discard.)

Example 2, high density: PCV = 0.21 mL

0.21 mL x 100 mL = 105.0 mL

0.20 mL

Therefore, add 5.0 mL of VBD to each 100 mL of cell suspension.

Check the adjusted cell suspension by centrifuging a 7.0 mL portion as 

before.

Note: Keep the cell suspension in the refrigerator when not in use. 

Always swirl the flask gently before use to secure an even 

suspension of the erythrocytes.

4.3.3 Preparation of hemoglobin color standards

Eleven color standards are used to read the hemolysin, complement, and antigen 

titrations. These standards are prepared by combining proportionate amounts 

(Illustration 2) of hemoglobin solution and 0.28% cell suspension as follows:

1. Preparation of hemoglobin solution

a. Pipette 1.0 mL of the 2.8% cell suspension into a large test tube 

(17 x 100-mm).

b. Add 7.0 mL DW and shake the mixture until all cells are lysed.
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c. Add 2.0 mL of the stock buffer solution (5X VBD) to restore 

tonicity and mix thoroughly.

2. Preparation of 0.28% cell suspension

Pipette 1.0 mL of the 2.8% cell suspension into a large test tube, add 

9.0 mL of VBD, and mix thoroughly.

3. Preparation of color standards

a. Label 11 serologic tubes (12 x 75-mm) with the specified % of 

hemolysis shown in Illustration 2. Mix the varying ratios of 

hemoglobin solution and the 0.28% cell suspension as shown.

ILLUSTRATION 2

Reagents Percent hemolysis 

0 10 20 30 40 50 60 70 80 90 100

Hemoglobin solution 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

0.28 % cells 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 

b. Shake the tubes and centrifuge at 600 X g for 5 minutes. Remove 

from the centrifuge without agitation. When not in use, store in 

refrigerator to prevent excessive change in color. The color standards, 

prepared for reading the complement titration, may be used the next 

day to read tests.

4.3.4 Hemolysin titration

1. Preparation of 1:100 hemolysin (rabbit antiserum against sheep 

red cells) solution

Thoroughly mix 98.0 mL of VBD and 2.0 mL of glycerinated hemolysin 

(lyophilized hemolysin with its glycerine containing diluent is equivalent). 

Dispense in convenient small aliquots and store at -20°C or lower. Do not 

refreeze after thawing.

2. Determination of hemolysin dilution needed for sensitization of 

2.8% sheep cells

Perform a hemolysin titration each time a new lot of 1:100 hemolysin 

solution is prepared and each time a new lot of sheep cells is used.
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a. Place 6 serologic tubes (12 x 75-mm) in a rack and label with 

hemolysin dilutions shown in last column of Illustration 3. Prepare 

dilutions for titration as shown. Thoroughly mix each dilution.

Note: These final hemolysin dilutions have been found appropriate 

for commercially prepared hemolysins. If they are not satisfactory, 

prepare other dilutions that suffice for the particular lot of 

hemolysin in routine use.

ILLUSTRATION 3

Preparation of Hemolysin Dilutions

Hemolysin

dilution, 1 mL Diluent, mL

Final hemolysin

dilution

1:100---------plus

1:1000-------plus

1:1000-------plus

1:1000-------plus

1:1000-------plus

1:1000-------plus

9.0--------gives

1.0--------gives

1.5--------gives

2.0--------gives

3.0--------gives

7.0--------gives

1:1000

1:2000

1:2500

1:3000

1:4000

1:8000

b. Label a second set of 6 serologic tubes with the correct hemolysin 

dilutions. Add 1.0 mL of the standardized 2.8% sheep cell suspension 

to each tube.

c. Add 1.0 mL of each of the final hemolysin dilutions (1:1000 

through 1:8000) to the 1.0 mL portions of sheep cell suspension with 

constant swirling of the contents. Incubate for 15 minutes at 37oC. 

After the incubation period, the sheep erythrocytes are “sensitized 

cells," ready for use.

d. Make a 1:400 dilution of guinea pig complement (C') by drawing 

up undiluted C' in a 1.0-mL pipette beyond the 0.6 mL mark. Wipe the 

tip of the pipette and return excess C' above the 0.6 mL mark to the 

stock container. Deliver 0.25 mL of C', without using the last 0.1 mL

of graduation of the pipette, into 99.75 mL of cold VBD. The diluted 

C' must be kept cold and be used within 2 hours.

Note: With less active C', it may be necessary to use a 1:300 

dilution, or with a more potent C', a 1:500 dilution may be needed 

for proper results.
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e. Label 6 serologic tubes with the final hemolysin dilutions. To each 

tube add 0.4 mL of cold VBD, 0.4 mL of the 1:400 C' dilution, and 0.2 

mL of cells sensitized with the series of final hemolysin dilutions 

(Step c) to the properly labeled tube. Mix and incubate in a 37°C 

water bath for 30 minutes. Centrifuge at 600 X g to pack the cells. 

Read the % hemolysis in each tube by comparing with the color 

standards.

f. Plot the amount of hemolysis obtained with each dilution of 

hemolysin on ordinary graph paper. Determine the optimal dilution of 

hemolysin from the graph by inspection. It is desirable to select a 

dilution such that further increase in hemolysin (as the curve 

progresses to the right) does not appreciably change the % lysis. The 

dilution selected also needs to provide for a slight excess of hemolysin. 

See Illustration 4.

Note: On the X-axis, let the left end of the hemolysin dilution scale 

be 0, and measure a suitable length for the 1:1000 dilution. Other 

dilutions are represented as fractions of this length. Thus 1:2000 = 

1/2 the length of 1:1000; 1:2500 = 2/5 of 1:1000; 1:3000 = 1/3 of 

1:1000; 1:4000 = 1/4 of 1:1000; and 1:8000 = 1/8 of 1:1000.

ILLUSTRATION 4

Hemolysin titration

Note: Increasing the hemolysin concentration (left to right) with 

complement held constant results in a "plateau" from which the 

optimal hemolysin dilution is selected. The optimal hemolysin 

dilution in this instance is 1:2000.

4.3.5 Preparation of sensitized cells 

1. Add 1 volume of optimal hemolysin dilution (example, 1:2000 dilution 

shown in Illustration 4) to 1 volume of the standardized 2.8% cell 

suspension with rapid, but gentle, swirling.

2. Before using, incubate for 15 minutes at 37°C.

4.3.6 Complement (C') titration

Note: For routine qualitative testing, the following procedure for handling 

complement has been found to be satisfactory: Commercially obtained 

lyophilized guinea pig complement is restored as directed. A 1:20 dilution of 

restored C' is made in 1X VBD; this dilution is dispensed in convenient 

(amount to be used at one time for tests) aliquots into inert plastic vials and 
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stored at -70°C or colder. The C' dilution is thawed (10°C water bath) and 

further diluted for titration. After the titer is determined, the remaining vials 

are thawed and diluted as needed for test use. A vial of each batch so 

prepared should be titered every 2 weeks to check for changes. There may be 

no significant decrease in titer over a period of several months.

Titration of C' is performed as follows:

1. Prepare sensitized cells.

2. Label 4 serologic (12 x 75-mm) tubes as in Illustration 5. To obtain 

accuracy, titrate in duplicate and use the average hemolysis.

3. Prepare 1:400 dilution of C'. (The 1:400 dilution of C' prepared 

previously and stored in the refrigerator may be used for titrations of 

complement and hemolysin when performed at the same time.)

4. Add reagents in the order shown in Illustration 5.

5. Shake tubes and place in the 37°C water bath for 30 minutes. Shake 

once at 15 minutes.

ILLUSTRATION 5

Complement Titration

Reagent Tube Number

1 2 3 4

VBD

1:400 dilution of C’

Sensitized cells

0.6 mL

0.2 mL

0.2 mL

0.55 mL

0.25 mL

0.2 mL

0.5 mL

0.3 mL

0.2 mL

0.4 mL

0.4 mL

0.2 mL

6. Remove tubes from water bath and centrifuge 600 X g to pack the 

cells. Determine the % hemolysis in each tube by comparing with the 

color standards, interpolating to the nearest 5% when a tube does not 

exactly match 1 of the standards.
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ILLUSTRATION 6

Conversion of % lysed cells, y, to ratio of % lysed to non-lysed cells, y/100-y

y y/100-y y y/100-y y y/100-y

10

15

20

25

30

35

0.111

0.176

0.25

0.33

0.43

0.54

40

45

50

55

60

65

0.67

0.82

1.0

1.22

1.50

1.86

70

75

80

85

90

2.33

3.00

4.0

5.7

9.0

7. Record the % hemolysis (y) for each tube. Using the chart in 

Illustration 6, determine the ratio of % lysed to non-lysed cells, y/100-y, 

for each volume (mL) of the 1:400 C’, as shown in the example below. 

The “y” values are hypothetical.

Example:

Tube # Vol (mL)of % Hemolysis Ratio

1:400 C’ (y) (y/100-y)

1 0.20 25 0.33

2 0.25 40 0.67

3 0.30 70 2.33

4 0.40 85 5.70

8. For each of the 4 tubes, plot on log-log graph paper the volume of the 

1:400 dilution of C' in mL against the ratio found in the chart. (Do not 

graph when "y" exceeds 90% nor when "y" is less than 10%.) Join the 2 

points plotted for the first 2 tubes and find the midpoint as shown in 

Illustration 7. Do the same for the last 2 tubes. (A good titration should 

have 2 points on each side of 1). Draw a line through the 2 midpoints. 

Draw a horizontal line through the intersection of this line with the vertical 

"I" line and read the C'H50 in mL of the 1:400 dilution of C'. Five C'H50

are required in the diagnostic test.
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In the example, C'H50 is 0.27 mL. Five units are contained in 1.35 mL (5 

X 0.27) of the 1:400 dilution. The dilution of C' necessary to obtain 5 

C'H50 in 0.4 mL is calculated as follows:

400 = X 1.35 X = 160

X = 118.5 or 

119

1.35 0.4

Hence 0.4 mL of a 1:119 dilution of C' contains 5 C'H50. (Direct readings 

for proper dilutions may be found in Illustration 8.)

Note: 0.4 mL of this working dilution of C’ contains 5 units of C'H50

when reacted with 0.2 mL sensitized 2.8% sheep red blood cells, as 

titrated above. 0.05 mL (50 μl) of this working dilution results in the 

same concentration (5 units of C'H50) when reacted with 0.025 mL

(25 μl) sensitized 2.8% sheep red blood cells, as used in the COFAL 

microtiter assay (Sections 4.3.7 and 4.4).

9. Determine the slope of the line joining the midpoints of the C’ 

titration plot. First, measure from any point near the left end of the line 10 

cm horizontally to the right. Then measure the vertical distance upward 

from the right end of the horizontal line to the slope line. Divide the 

vertical measurement by 10 cm to obtain the slope (1/N). The normal 

value for a valid slope is 0.20 ± 10%. 
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ILLUSTRATION 7

Graphing Method for Complement Titration

[use log-log paper]

y axis = mL volume of 1:400 dilution of C’

x axis = the ratio “%Lysed / %non-lysed cells” (y/100-y)
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ILLUSTRATION 8

Complement Dilutions for Conversion of 1 C'H50 to 5 C'H50s in the LBCF Test

ML C’ -

1 C’H50

For 5 C’H50s, Dilute Stock C’

1:300 C’ 1:350 C’ 1:400 C’ 1:450 C’ 1:500 C’

0.21 114 133 152 171 190

0.22 109 127 145 164 182

0.23 104 122 139 157 174

0.24 100 117 133 150 167

0.25 96 112 128 144 160

0.26 92 108 123 138 153

0.27 89 104 119 133 148

0.28 86 100 114 129 143

0.29 83 97 110 124 139

0.30 80 93 107 120 133

0.31 77 90 103 116 129

0.32 75 87 100 113 125

0.33 73 85 97 109 121

0.34 71 82 94 106 118

0.35 69 80 91 103 114

4.3.7 Serum and antigen titrations

The serum is a standard antiserum for the test, and the antigen is a known gs 

antigen-containing reagent. To conserve serum, the titrations are done in a 

microtiter block test system.

1. Preparation of serum for inactivation

a. Make an initial dilution (in 1X VBD) of positive serum which will 

be fourfold lower (4X) than the stated titer (if known or estimated).

b. Inactivate the diluted serum for 30 minutes at 56°C.

c. The diluted and inactivated antiserum may be dispensed into 

convenient small aliquots (sufficient for a day's test) and stored at 

-70°C or lower.
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2. Addition of VBD to plates (see Illustration 9)

a. Add 25 μL (0.025 mL) VBD to the wells in which the antigen 

dilutions are to be made, except the first one in each row. (In 

Illustration 9 this includes wells 2 to 8, A through H). Also add 25 μL

to the 3 wells of the serum-complement controls (9A through 9C) and 

to the 5 wells of serum control (9D through 9H).

b. Add 50 μL (0.05 mL) VBD with a pipette dropper to each of the 3 

wells of the VBD complement controls (10A through 10C)

c. Add 100 μL (0.1 mL) VBD with a pipette dropper to a well for the 

cell control (10H).
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ILLUSTRATION 9 - Template for serum and antigen titrations

Anticomplementary

Controls

1 2 3 4 5 6 7 8 9 10 11 12

1.25 C’H50 A Serum 

(4X) C’ 

Control

VBD 

Control x

0%

2.5 C’H50 B Serum 

(4X) C’ 

Control

VBD 

Control x

10%

5.0 C’H50 C Serum 

(4X) C’ 

Control

VBD 

Control x

20%

Serum dilutions

X

4

D

X/4 

serum 

control x x

30%

X

2

E X/2 

serum 

control x x

40%

X F

X serum 

control x

100% 50%

2X G 2X 

serum 

control x

90% 60%

4X H 4X 

serum 

control

Red cell 

control

80% 70%

1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256 Hemolysis

Standards

Antigen Dilutions
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3. Addition of antigen to plates

a. Add 25 μL of the test antigen to the first 2 wells of each row (1 

and 2, A through H, Illustration 9).

b. Using a pipetter set at 25 μL (or a 0.025 microdiluter), mix and 

make serial twofold dilutions starting with wells 2A through 2H and 

continuing to wells 8A through 8H. Discard the 25 μL after mixing the 

number 8 wells.

4. Addition of serum to plates

a. Make serial twofold dilutions in tubes of the serum prepared in 

Step 1. These dilutions will be 4X (the concentration as prepared in 

Step 1), 2X, X (the expected titration endpoint), X, and X.2 4

b. Add 25 μL of the corresponding dilution of serum to the wells as 

shown in Illustration 9. The dilutions will be added to wells 1 through 

9 in the appropriate row (rows D-H). The 4X concentration will also 

be added to wells 9A through 9C.

c. Shake the plates and allow to stand at 4°C until the complement is 

added. Stack or cover plates to prevent evaporation.

5. Preparation and addition of complement

a. Make the dilution of complement which will contain 5 C'H50 in 0.4 

mL (see Section 4.3.6). From this, make a 1:2 and a 1:4 dilution to 

give 2.5 and 1.25 units, respectively.

b. Add 50 μL of this complement dilution to all wells getting 5 units 

of complement (wells 1-9, rows C through H in Illustration 9).

c. Add 50 μL of a 1:2 dilution of complement to all wells getting 2.5 

units of C' (wells 1-9, row B).

d. Add 50 μL of a 1:4 dilution of complement to all wells getting 

1.25 units of C' (wells 1-9, row A).

e. Shake the plates and incubate at 4°- 6°C for 15 to 18 hours. Stack 

or seal plates to avoid evaporation.
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6. Addition of sensitized red cells

a. Add 25 μL of sensitized sheep red blood cells to all the wells. 

Shake immediately, tape, and incubate at 37°C.

Note: Incubation may be done in a water jacketed incubator, or by 

floating the plates in a 37°C water bath for 30 minutes. The plates 

should be shaken gently every 10 minutes to keep the cells 

(remaining) in suspension or the plates may be placed on a 

mechanical shaker in a 37°C incubator for the 30 minutes period. 

A shaker with a reciprocal action traversing a course of 2-3 mm at 

approximately 250-500 times per minute will keep the cells in 

suspension and not cause splashing. More vigorous shaking must 

be avoided to prevent mechanical lysis of cells or inactivation of 

complement.

b. After incubation, add 125 μL (0.125 mL) of each color standard to 

the plates. 

c. Centrifuge plates at 600 X g for 5 minutes or allow cells to settle at 

4°C for 4 to 6 hours.

d. Read by comparison with color standards. Record % hemolysis on 

record sheets and determine optimal dilution of antigen and titer of the 

serum. Use 4 units of antiserum for each test.

4.4 Detection of gs antigen in test material 

Store all test samples, including positive and negative controls, at -60°C or colder until 

the CF test is conducted. Just prior to testing, thaw and refreeze each sample (test sample 

and control samples) 3 times to disrupt intact cells and to release group specific antigen, 

if present.

The antiserum used in these tests is one produced in rabbits against the P-27 antigen of an 

avian leukosis virus. This reagent will be supplied by the Center for Veterinary Biologics

(CVB).

4.4.1 Test wells

Add reagents in the following order: (See Illustration 10)

1. Add 25 μL VBD to all test wells, rows A-H, wells 1-9.
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2. Using a pipetter set at 25 μL (or a 0.025-mL microdiluter), add 

appropriate antigen suspension to the first well of each test series and 

make the 1:2, 1:4, 1:8 dilutions. Change the pipette tips (or blot the 

diluter) and pick up another aliquot of antigen to make the 1:2 anticomplementary test dilution.

3. Add 25 μL of 4X or 4 unit antiserum to all test wells receiving 

antiserum (not the anti-complementary test wells). Add 25 μL for VBD to 

the anti-complementary test wells.

4. Shake plates and stack to prevent evaporation until complement is 

added.

5. Prepare working dilution of C’ as determined in Section 4.3.6. Add 

50 μL to all the test wells (rows A-H, wells 1-9). This is best done late in 

the afternoon. Shake plates and incubate at 4°C for 15 to 18 hours. Stack 

or cover to prevent evaporation.

6. Add 25 μL of the prepared presensitized red cells to all wells. Tape 

and seal plates. Place on a slow speed reciprocal shaker at 37°C and 

agitate constantly (with just enough force to keep the cells from settling) 

for 30 minutes. Remove from incubation and centrifuge at 600 X g for 5 

minutes.

7. Read by comparison with color standards and record % hemolysis. 

4.4.2 Control wells

Only 1 set of controls is needed for each group of tests. These additions are made 

at the same time as the test wells.

1. In Row 12, add VBD to the following: 25 μL to wells A and B’, 50 μL

to wells C, D, and E’, and 100 μL to well F.

2. Add 25 μL antiserum (4 units) to wells 12A and 12G.

3. Add 25 μL of a positive antigen control (4 units) to wells 12B and 

12G.

4. Add 50 μL working dilution complement to wells 12A, 12B, 12C, and 

12G. Dilute complement 1:2 and add 50 μL to well 12D. Dilute 

complement 1:4 and add 50 μL to well 12E.
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5. Add 25 μL sensitized red cells to all wells and proceed as in the

screen- ing tests. For samples of results of completed test see Illustration 

11.

ILLUSTRATION 10

Template for CF plate

1 2 3 4 5 6 7 8 9 10 11 12

A 1:2 X0% Serum

A/C

B 1:4 X10% Pos. Ag

A/C

C 1:8 X20% 5

C’H50

D A/C

1:2 X

30% 2.5

C’H50

E 1:2 X40% 1.25

C’H50

F 1:4 100% 50% Red cell

control

G 1:8 90% 60% Pos. Ag

Pos. Serum

H A/C

1:2

80% 70%

 X

 Color Stds
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ILLUSTRATION 11 - Sample plate readings

N-1

1

N-2

2

N-3

3

S1-1

4

S1-2

5

S1-3

6

S2-1

7

S2-2

8

S2-3

9 10 11 12

A

1:2

100 100 100 0 0 0 100 100 100 X0% 100 Serum

A/C

B

1:4

100 100 100 30 0 0 100 100 100 X10% 100 Pos. Ag.

A/C

C

1:8

100 100 100 70 10 0 100 100 100 X20% 100 5 

C’H50

D

A/C

1:2

100 100 100 100 100 100 100 100 100 X30% 100 2.5 

C’H50

E

1:2

100 30 20 0 0 0 0 0 0 X40% 50-

70

1.25

C’H50

F

1:4

100 80 40 0 0 0 0 0 0 100% 50% 0 Red cell

control

G

1:8

100 100 100 20 0 0 30 0 0 90% 60% 0 Pos. Ag. 

Pos. Serum

H

A/C

1:2

100 100 100 100 100 100 100 100 100 80% 70% X

S3-1 S3-2 S3-3 R1-1 R1-2 R1-3 R2-1 R2-2 R2-3 Color stds

N = uninoculated culture S = test sample culture

R = positive control culture Number in square = % hemolysis
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5. Report of Test Results

Presence of complement-fixing activity in the harvested samples (from passages) at the 1:4 or 

higher dilution, in the absence of anticomplementary activity, is considered positive unless the 

activity can definitely be established to be caused by something other than infectious lymphoid 

leukosis virus. Activity at the 1:2 dilution is considered to be suspicious, and the sample must be 

further subcultured to determine the presence or absence of the group-specific antigen. 

5.1 Record the results as percentages for each dilution of each passage, and report the 

final results of each test as negative/satisfactory or positive/unsatisfactory.

5.2 Report "no test" in case of reagent or control failure and specify which.

6. Repeat Tests

The decision to repeat a test will be made according to the merits of each situation.

7. References

7.1 Sarma, P. S., H. C. Turner, and R. J. Huebner. An avian leukosis group specific 

complement-fixation reaction. Application for the detection and assay of noncytopathogenic leucosis viruses. Virology 23:313-321 (1964).

7.2 Public Health Monograph No. 74. Standardized diagnostic complement fixation 

method and adaptation of micro test (1965). U.S. Department of Health, Education and 

Welfare, Washington, D.C. 20201.

7.3 A Guide to the Performance of the Standardized Diagnostic Complement Fixation 

Method and Adaptation to Micro Test. 1st Edition, July 1, 1969. U.S. Department of 

Health, Education, and Welfare, Washington, D.C. 20201.

7.4 Sever, J. L. Application of a microtechnique to viral serological investigations. J. 

Immunol. 88:320-329 (1962).

7.5 Title 9, Code of Federal Regulations, section 113.31, U.S. Government Printing 

office, Washington, DC.
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8. Summary of Revisions

Version .04

 The Contact information has been updated; however, the Virology Section has elected 

to keep the same next review date for the document.

Version .03

 The contact information was updated.

Version .02

This document was revised to clarify the practices currently in use at the Center for Veterinary 

Biologics and to provide additional detail. While no significant changes were made that impact 

the outcome of the test, the following changes were made to the document:

 The document number has been changed from PYSOP0405 to SAM 405.

 The Contact information has been updated.

Version .01

This document was completely revised and rewritten in new format to 1) meet the current 

NVSL/CVB QA requirements, 2) update the reagent and equipment lists, 3) allow for alternate 

methods of cell culture preparation and handling, 4) include treatment methods for additional 

vaccines, and 5) to provide additional testing clarification. No significant changes were made 

from the superseded protocol. 
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Appendices: Counting Cells in Suspension with a Neubauer Hemacytometer and 

Formulating Cell Suspensions

The protocol in this appendices describe a method to count cells in suspension with the use of a 

1/10 mm deep Neubauer hemacytometer and microscope. It also explains how to formulate cell 

suspensions for specific concentrations and volumes.

Appendix I

Materials

1. Equipment/instrumentation

Equivalent equipment or instrumentation may be substituted for any brand name listed below.

1. Hemacytometer (1/10 mm deep Neubauer hemacytometer)

2. Incandescent microscope (capable of 100X magnification

3. Hand Tally Counter (Daigger Cat. No. GX6594)

2. Reagents/supplies

Equivalent reagents or supplies may be substituted for any brand name listed below.

1. Trypan Blue (0.4%)

2. Snap-cap tube, 6-mL, 12 x 75-mm, (Falcon, Cat. No. 2058)

3. Disposable plastic pipettes, 1-, 5-, 10-, and 25-mL

4. Pipette (Labsystems, Model Finnpipette Digital 5- to 40-μL)

5. Disposable pipette tips
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Appendix II

Counting cells with a hemacytometer

In a 6-mL snap-cap tube, dilute 0.5 mL of the cell suspension to be counted with 1.0 mL of 0.4% 

Trypan Blue solution. Replace the cap on the tube and mix the suspension by inverting the tube 

several times. Place the cover slip over the counting grid on the hemacytometer. While the cells 

are still suspended, quickly load the chamber of the hemacytometer with this preparation using a 

pipette. Fill the chamber with enough of the preparation to completely cover the grid under the 

cover slip. Overfilling can cause erroneous counts. The total volume of cell suspension in 10 

1-mm squares (see numbered squares) under the cover slip is equal to 1 cubic mm or 1 μL.

1 2

5

4 3

6 7

10

9 8

Figure 1.

Let the preparation stand for approximately 1 minute to allow the cells to settle onto the bottom 

of the counting chamber. Examine the grid with a microscope under 100X magnification. Check 

for the even distribution of cells. (Any irregularity of distribution will cause erroneous results.) 

Count the live cells (dead cells stain blue while live cells remain translucent). Count only those 

live cells that fall on the top line, the left line, or within each numbered 1-mm square (1-10) in 

the grid (Figure 1). Keep track of the total number of cells counted for the 10 squares with a 

Hand Tally Counter. 

Calculate the number of cells per ml in the initial suspension by multiplying the total cell count 

by the dilution in Trypan Blue (3) and by the conversion factor from cubic mm μL to mL (1000). 

See example below.

Example:

number of cells in 10 squares= 385

 1:3 cell dilution= x 3

 1155

cubic mm/mL x 1000 

number of cells per mL = 1155000
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Appendix III

Formulating cell suspensions

1. First calculate the Dilution Factor (DF) by dividing the concentration of cells/mL in the 

initial suspension by the desired final concentration of cells/mL. 

Example:

If the concentration of cells in the initial suspension is 1155000 cells/mL and the desired final 

concentration is 350000 cells/mL, then: 

 DF = 1155000 cells/mL divided by 350000 cells/mL = 3.3 

2. Next, divide the desired final volume of cells by the DF. The result of this division will tell 

you the volume of the initial cell suspension you will need to make the final desired volume of 

cells. 

Example: 

If the final desired volume of cells is 375 mL, then:

375 mL/3.3 (DF) = 113.6 mL

This is the volume of the initial cell suspension needed to make the final volume of cells. (Round 

this number to 114.)

3. Finally, take the amount of the initial cell suspension determined in Step 2 and add sufficient 

media to bring it to the final volume of cells desired. 

Example:

Add 261 mL (375 mL - 114 mL) of media to 114 mL of the initial cell suspension (1155000 

cells/mL) to get 375 mL of the desired 350000 cells/mL suspension.