drug discovery tools for immunotoxicology research

Drug Discovery Tools for Immunotoxicology Research

Table of Contents

Immunotoxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3

Immunotoxicology Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-5

Immunosuppression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3

Clinical Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3

Blood Cellular Levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3

Cell Phenotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3

Immune Function Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3

Immunogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4

Hypersensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4

Type I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4

Type II/III . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4

Type IV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5

Pseudoallergy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5

Autoimmunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5

Adverse Immunostimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5

Drug Discovery Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6

Micronucleus Analysis Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6

BrdU Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6

Rat Immunophenotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7

Dog Immunophenotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9

Non-Human Primate Immunophenotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10

BD Cytometric Bead Array (BD CBA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13

Reagents for Immunotoxicology Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15

Micronucleus Analysis Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15

BrdU Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15

Rat Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15

Dog Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22

Non-Human Primate Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25

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Over the past decade, drug discovery research has expanded toinclude a battery of new assays to identify potentially immunotoxiceffects of xenobiotics (drugs/chemicals that are not native to themammalian system). This field of research is known asimmunotoxicology: the study of adverse health effects resultingfrom xenobiotic interactions with the immune system.1 In October2002 the US Food and Drug Administration (FDA) publishedguidelines to define the major areas of immunotoxicologyresearch and to discuss assays that are available to determinethese effects. Although we focus on the US FDA guidelines,similar guidelines are in place in several countries worldwide.

BD Biosciences is committed to supporting the drug discovery processby providing high-quality reagents for immunotoxicology screening.

To download the complete US FDA guidelines, Guidance forIndustry: Immunotoxicology Evaluation of Investigational NewDrugs, visit www.fda.gov/cder/guidance/4945fnl.doc

Immunotoxicology OverviewDuring the development of an investigational new drug, it isimportant to assess the potential adverse effects the drug will haveon patients. Prior to clinical testing of a drug candidate, toxicologystudies are conducted in laboratory animals, primarily rats, dogs,and non-human primates.

These assays routinely involve a 28-day, repeat-dose study, withadjustments for potential stress-related immune effects due tochanges in the animals’ environment at the onset of the study. Twotypes of testing strategies that can be used in immunotoxicologystudies include identifying obvious, basic changes to the immunesystem composition and structure, and in vitro testing that leadsto the identification of the potential mechanism of the negativeeffect. The identification of changes within the first type of testingstrategy may require additional testing of a more complex nature.When beginning an immunotoxicity study, some considerations arethe type of administration (such as topical, inhalation, or injection)and the condition of the potential patient (such as treating animmunosuppressed patient or pregnant woman). These considerationswill help define what type of toxicology testing is required orsuggested.

There are five major areas of immunotoxicology as defined by theUS FDA guidelines: immunosuppression, immunogenicity,hypersensitivity, autoimmunity, and adverse immunostimulation.

Immunosuppression

Immunosuppression occurs as an unintended impairment of anyimmune component or function. Immunosuppression can beidentified by a drop in blood cell numbers, a decrease in theamount of circulating immunoglobulin (Ig) levels, alterations inthe histology of immune tissues, and an increased potential forinfection and/or tumor development.2 The following is a list oftools to detect immunosuppressive effects.

1. Chemistry values and histopathology results routinely providethe first evidence that a drug has an immunological effect.3

These can include changes in blood cell counts, immune organweight, and architectural changes to immune tissues (spleen,thymus, lymph nodes, bone marrow, and any lymphoid tissuethat drains the site of drug administration). An additionalassay recommended by the FDA because of its ease of use isthe quantitation of serum Ig levels.

2. Changes in blood cellular levels can be due to a variety ofcauses. To identify changes based on bone marrow toxicity orloss of progenitor development, researchers use assays suchas the granulocyte-macrophage colony forming unit (CFU-GM)clonogenic assay.4 Drug-mediated hemolysis is usually identifiedby an increase in white cell counts, increased spleen weight,iron deposition, and reticulocytosis.5 In vitro detection ofhemolysis can be done by incubating the drug candidate witherythrocytes and determining the release of hemoglobin.6

3. If initial studies identify signs of immunosuppression, it isrecommended that additional tests be conducted to determinethe potential mechanism. Cell phenotyping and enumerationis an easy and powerful tool to quickly identify changes inspecific leukocyte levels. A summary of markers used toidentify different cell types by flow cytometry is shown onpage 4, Table 1.

4. Immune function studies are critical for understanding themechanisms of immunosuppression. Depending on the responsesseen in initial studies, there are several assays that are commonlyused to dissect and better understand immunosuppressivechanges.7

a. The T cell-dependent antibody response is the most widelyaccepted method. The anti-sheep red blood cell assay(plaque assay) has been used successfully and extensively.Modifications of this assay to detect both IgM and IgGresponses utilizing ELISA and ELISPOT techniques arealso effective.8

b. Natural Killer (NK) cell function assays identify the abilityof NK cells to kill tumor cells in vitro. The most commonmethods include chromium release cytotoxicity assays orflow cytometry-based assays.9, 10

Immunotoxicology

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c. In vitro blastogenesis assays determine the ability oflymphocytes to respond to mitogens such as Concanavalin Afor T cells or Lipopolysaccharide for B cells.11,12 In addition,signaling through cell receptors can be assessed by usingmonoclonal antibodies to stimulate cells. Detection assaysinclude tritiated thymidine incorporation, MTT detection,Granzyme B detection, and BrdU incorporation.13

d. Cytotoxic T cell (CTL) assays demonstrate the ability ofCTLs to kill target cells. These assays include CTL killingof tumor cells or a mixed leukocyte reaction assay.14. 15

e. Changes in cytokine profiles can also be indicative of animmunotoxic effect. Proinflammatory and immunoregulatorycytokines (Th1/Th2) are commonly measured.16

f. Suppression of contact delayed hypersensitivity response isa useful indicator of immunosuppression.17

g. Host resistance assays determine the ability of a laboratoryanimal to resist either laboratory infection or a transplantabletumor after administration of a drug candidate.1 Whilethese assays are cumbersome and expensive, they areexcellent for assessing immunosuppression using an in vivo model.

It is important to remember that some drugs are designed to induceimmunosuppression and thereby distinguishing between unintendedand intended immunosuppression is crucial. It has been found thattwo or three immune parameter alterations are required to identifya hazard. The measurement of T cell-dependent antibody responsealong with phenotypic characterization is particularly useful.1

Immunogenicity

The term immunogenicity in this context refers to the ability of adrug to induce an immune response. The level of immunogenicitycan depend on several factors, but the crucial factor for drugimmunogenicity is size. Most low molecular weight compounds(1000 – 5000 Mr) do not elicit an immune response by themselves.However, when they covalently bind to proteins, they can becomehapten-protein complexes that can be antigenic. High molecularweight compounds (>10,000 Mr) are more likely to be antigenicand can be evaluated in animal models prior to clinical testing.Typical in vitro assays for determining an anti-drug immuneresponse include ELISAs for anti-drug antibodies and standardin vitro blastogenesis assays described above.

There are two main concerns when evaluating immunogenicity.First, whether the immune response illicited will affect thebiological activities of the drug. Second, whether the drug willinduce a drug allergy or hypersensitivity response.

Hypersensitivity

Hypersensitivity refers to an adverse immunological reaction inresponse to the drug given (ie, drug allergy). There are four classesof hypersensitivity, each unique in its mechanism and response.

1. Type I – immediate-type hypersensitivity (IgE-mediated) canhave 2 subtypes of reactions: systemic hypersensitivity orrespiratory hypersensitivity. An important consideration forassessing this type of reaction is the route of administeringthe drug candidate. Three common tests for this type ofreaction are the passive cutaneous anaphylaxis assay (PCA),the active cutaneous anaphylaxis assay, and the active systemicanaphylaxis assay (ASA) in the guinea pig. These assays areconsidered limited, as they do not correlate directly withreactions in humans. Mouse assays have been used to identifyrespiratory allergens including the mouse IgE test (MIGET),detection of serum cytokine pattern modulation, and themurine local lymph node assay (LLNA).18, 19, 20 The popliteallymph node assay (PLNA) is useful for detecting drug specificsystemic hypersensitivity.21

2. Type II and III – antibody-mediated cytotoxic reactions (IgG-or IgM-mediated) are due to IgG and/or IgM antibody responsesto drugs and drug metabolites. Type II pathologies are due toantibody-dependent cellular cytotoxicity (ADCC) and/orcomplement-mediated lysis of somatic cells. Type IIIpathologies are due to immune complex formation, deposition,and complement activation with local tissue destruction. Thesetwo types of reactions are seldom modeled in animals, andthere are no standard assays for assessing Type II or IIIhypersensitivity.

Immunotoxicology Overview (continued)

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Not for use in diagnostic or therapeutic procedures. Not for resale.

Table 1. Cell Phenotype Markers

T cell marker

T cell subset marker

T cell subset marker

B cell marker

NK cell marker

Monocyte marker

Granulocytes marker

T cell activation marker

B cell activation marker

Adhesion receptor

Adhesion receptor

CD3 1F4 Figure 7

CD4 OX-38, OX-35 Figures 5,8

CD8a OX-8 Figure 5

CD45RA OX-33 Figure 4

NKR-P1A 10/78 Figure 7

CD11b WT.5 Figure 8

Granulocytes HIS48 Figure 9

CD25 OX-39 Figure 6

CD80 3H5 Figure 4

CD29 Ha2/5 Figure 10

CD62L HRL1 Figure 11

Pan T LSM8.358 Figure 13

CD4 LSM12.125 Figure 13

CD8 LSM1.140 Figure 13

Pan B LSM11.425 Figure 12

NK cell marker DL10*

CD11b CA16.3E10*

CD11b CA16.3E10*

Activation marker CTL 2.58 Figure 14

Activation marker CTL 2.58 Figure 14

CD49d CA.4.5B6*

CD54 CL18.1D8*

CD3 SP34.2 Figure 18

CD4 M-T477 Figure 19

CD8 RPA-T8 Figure 20

CD20 2H7 Figure 17

CD56 MY31 Not shown

CD14 M5E2 Not shown

CD11b ICRF44 Figure 22

CD25 M-A251 Figure 21

CD86 2331(FUN-1) Not shown

CD29 MAR4 Not shown

CD62L SK11 Not shown

SPECIFICTY CLONE FIGURE SPECIFICTY CLONE FIGURE SPECIFICTY CLONE FIGURE

Rat Dog Non-Human Primate

* Not available from BD Biosciences

3. Type IV – delayed-type hypersensitivity (T lymphocyte-mediated) occurs as a skin reaction. Nonclinical studies toassess type IV responses have classically been conducted inguinea pigs using sensitization and challenge. Several FDA-accepted tests have been developed to assess a drug’s ability toinduce sensitization, including the Buehler assay, the guineapig maximization test, the split adjuvant technique, and theDraize test. Additionally, tests have been developed in mice,such as the mouse ear swelling test (MEST), that yield resultssimilar to the guinea pig assays.22 To detect the inductionof delayed-type hypersensitivity reactions, one techniquein particular has been used extensively with drugs knownas sensitizers. This assay, the LLNA, can be used as analternative to the guinea pig assays.23

4. Pseudoallergic (Anaphylactoid) reactions occur when theinflammatory or anaphylactic systems are activated, but notin response to a specific antigen. Anaphylactoid reactions aredifferent from anaphylaxis (IgE-mediated) and can bedifferentiated by mast cell histamine release and activation of the complement system.24 Detection of pseudoallergicresponses includes assays to detect serum complementproteins by ELISA or other related methods.


Autoimmunity

Autoimmunity occurs when the immune system fails to distinguishbetween self and non-self, producing antibodies directed againstits own tissue and activating an immune response to self antigens.This type of a response can cause tissue damage, immune complexdeposition, inappropriate activation of cells, and hypersensitivityreactions. Autoimmune reactions can be organ-specific or non-specific with local or systemic responses.

No standard assays have been validated for determining potentialautoimmune reactions. The most commonly used method is thePLNA. 21, 25 Adaptations of the LLNA have also been described toassess potential autoimmune reactions.23

Adverse Immunostimulation

Adverse immunostimulation refers to unregulated and unintendedactivation of some component of the immune system. This causeschronic inflammation and an infiltration of leukocytes in tissues.26

This type of reaction is not commonly seen with administration ofa drug candidate. In fact, chemicals with this type of activity areoften evaluated as potential adjuvants. There are no standardizedmethods for assessing this type of drug effect.

References

1. Toxicologic Pathology, 2002. 30(1): p. 54-58.

2. Toxicology Letters, 1998. 102-103: p. 267-270.

3. Toxicology, 1994. 86(3): p. 187-212.

4. Cell Biol Toxicol, 2001. 17(2): p. 95-105.

5. Toxic Responses of The Blood. Casarett & Doull's Toxicology. 2001, NewYork: McGraw-Hill. 389-417.

6. Red blood cell lysis (Basic Protocol 4). Current Protocols in Toxicology.Vol. 1. 1999, New York: John Wiley & Sons. 2.4.4.

7. Toxicology Letters, 1998. 102-103: p. 247-255.

8. Toxicology, 2000. 156(1): p. 1-11.

9. Journal of Immunological Methods, 2001. 253: p. 177-187.

10. Cytometry, 2000. 41: p. 289-297.

11. Fundam Appl Toxicol, 1993. 21(4): p. 535-545.

12. Toxicology, 1995. 96(2): p. 147-156.

13. Methods, 1999. 19: p. 28-35.

14. Toxicology, 1998. 127(1-3): p. 223-232.

15. Toxicology, 1997. 119: p. 95-101.

16. Methods, 1999. 19: p. 17-27.

17. Journal of Immunology, 2000. 165: p. 2374-2381.

18. Fundamental and Applied Toxicology, 1996. 33: p. 1-10.

19. Toxicology, 2001. 158: p. 51-58.

20. Toxicology and Applied Pharmacology, 1997. 145: p. 218-229.

21. Toxicology, 2001. 158: p. 65-69.

22. Toxicology and Applied Pharmacology, 1986. 84: p. 93-114.

23. Toxicology, 2001. 158: p. 59-64.

24. Crit Rev. Therapeutic Drug Carrier Syst, 2001. 18(6): p. 567-606.

25. Environ Health Perspect, 1999. 107: p. 673-677.

26. Eur J Nucl Med, 2001. 28(9): p. 1384-1393.

The micronucleus assay is an established method for studying in vivo chromosomal damage.It is based on the observation that displaced chromatin, resulting from chromosome lossor breakage, may fail to be incorporated into daughter nuclei as a cell divides. The resulting“micronucleus” is found in the cytoplasm. Elevations in the frequency of micronuclei (MN)are indicative of genotoxic activity.

The µicroFlow® and µicroFlowPLUS Micronucleus Analysis Kits are designed to measureMN events in peripheral blood cells of mice or rats. During erythropoiesis, an erythroblastexpels its main nucleus to become a reticulocyte (RET), while the MN remain in the cytoplasm.The newly formed RET is then released from the bone marrow into the circulatingbloodstream, where it develops into a normochromatic erythrocyte (NCE). µicroFlow®and µicroFlowPLUS are based on a RET-specific cell surface marker, CD71, whichdifferentially labels immature erythrocytes. When fixed peripheral blood cells areappropriately treated, NCEs and RETs with and without MN are easily resolved andquantified by flow cytometry. These kits measure the frequency of each cell population of interest (%RET, %MN-RET, %MN-NCE).µicroFlow® is a registered trademark of Litron Laboratories

Rat Immunophenotyping



µicroFlow® and µicroFlowPLUS Micronucleus Analysis Kits for Quantitative Analysisof Micronuclei by Flow Cytometry

BrdU Analysis for Detection of Proliferating Cells

The immunofluorescent staining of incorporated bromodeoxyuridine (BrdU) and flowcytometric analysis provide a high resolution technique to determine the frequency andnature of individual cells that have synthesized DNA. In this method, BrdU (an analog ofthe DNA precursor, thymidine) is incorporated into newly synthesized DNA by cells enteringand progressing through the S (DNA synthesis) phase of the cell cycle. The incorporatedBrdU is stained with specific anti-BrdU fluorescent antibodies. The levels of cell-associatedBrdU are then measured by flow cytometry.

Propidium Iodide

Reticulocytes

Parasitized RBCs

Nucleated

100 101 102 103 104

FITC

an

ti-C

D71

(O

X-2

6)

100

101

102

103

104A.

PE a

nti

-IL-

4 (O

X-3

1)

APC

an

ti-B

RD

U (

3D4)

APC anti-BRDU (3D4) APC anti-BRDU (3D4) APC anti-BRDU (3D4)7AAD

FITC

an

ti-I

FN-γ

(DB

1)

PE a

nti

-CD

25 (

OX

-39)

100 101 102 103 104100

101

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104

0 200 400 600 800100

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A. B. D.C.

B.

Figure 1B. Mouse erythrocytes stained withµicroflow reagent.

Figure 1A. FITC and PI staining of rodenterythrocytes for flow cytometric analysis

Figure 2. Multicolor flow cytometric analysis of proliferating (BrdU positive) rat cells that produce IFN-γ. Spleen cells from a rat were primed in vitrowith anti-CD3 and restimulated with PMA and ionomycin in the presence of a protein transport inhibitor (to promote intracellular cytokine accumulation).During the final 45 minutes of culture, the cells were labeled with 20 µM BrdU. The cells were harvested and stained with FITC anti-IFN-γ (DB1) (A),APC anti-BrdU (3D4), 7-AAD (B), PE anti-CD25 (OX-39) (C), and in a separate well PE anti-IL-4 (OX-31) (D).

Drug Discovery Tools


B lymphocytes

T lymphocytes

FITC anti-CD45RA (OX-33)100 101 102 103 104

PE a

nti

-CD

80 (

3H5)

100

101

102

103

104B.

anti-Marginal Zone B Cell marker (HIS57) on paraffin-embedded spleen

B.

100 101 102 103 1040

40

80

120

160

200

Cel

l Nu

mb

er

FITC anti-CD25 (0X-39)

anti-CD4 (OX-38) on frozen spleen

C.

FITC anti-CD4 (OX-35)100 101 102 103 104

APC

an

dt-

CD

3 (1

F4)

100

101

102

103

104D.

anti-CD8a (OX-8) on paraffin-embedded spleen

A.

100 101 102 103 104100

101

102

103

104

FITC anti-CD8a (OX-8)

APC

an

ti-C

D3

(1F4

)

B.

FITC anti-IgM (G53-239)100 101 102 103 104

PE a

nti

-CD

45R

A (

OX

-33)

100

101

102

103

104A.

100 101 102 103 104100

101

102

103

104

FITC anti-CD45RA (OX-33)

PE a

nti

-CD

80 (

3H5)

A.

Figure 4. Activation-induced changes in the level of expression of the CD80antigen on fresh rat splenocytes (A) and cells treated in vitro for 72 hours withlipopolysaccharide (LPS) (B). Cells were stained simultaneously with FITC anti-CD45RA (OX-33) and PE anti-CD80 (3H5).

Figure 3. Flow cytometric analysis of rat splenocytes using PE anti-CD45RA(OX-33) and FITC anti-IgM (G53-239) (A) and immunohistochemical stainingof rat spleen with anti-marginal zone B cell marker (H1S57) (B).

Figure 6. Expression of CD25 (IL-2Rα chain)on rat spleen T cells (CD3+) following invitro incubation for 48 hours withmedium alone (red line) or activation for48 hours with Concanavalin A (green line).

Figure 5. Immunohistochemical staining of rat tissue with anti-CD8a (A) or anti-CD4 (C) and flow cytometric analysis of rat peripheral blood cellsstained with the APC anti-CD3 (1F4) and FITC CD8a (OX-8) (B) or anti-CD4 (OX-35) (D).

Rat Immunophenotyping (continued)



100 101 102 103 104100

101

102

103

104

FITC anti-CD3 (1F4)

PE a

nti

-NK

R-P

1A (

10/7

8)

NK-T Cells

NK Cells

100 101 102 103 104

APC

an

ti-C

D3

(1F4

)

PE anti-CD4 (OX-35)

CD4- T cellsCD4+ T cells

CD3- CD4+Monocytes

100

101

102

103

104A.

FITC anti-CD11b (WT.5)100 101 102 103 104

Cel

l Nu

mb

er

0

10

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30

B.

Forward Scatter0 50 100 150 250

Sid

e Sc

atte

r

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Granulocytes

Lymphocytes/ Monocytes

A.

APC anti-Granulocyte Marker (HIS48)100 101 102 103 104

Cel

l Nu

mb

er

0

60

120

240

300

180

B.

anti-CD29 (Ha2/5)

A.

100 101 102 103 104100

101

102

103

104

FITC anti-CD29 (Ha2/5)

PE a

nti

-CD

3 (1

F4)

B.

100 101 102 103 104100

101

102

103

104

FITC anti-CD62L (HRL1)

PE a

nti

-CD

3 (1

F4)

Figure 7. Rat peripheral blood leukocytesstained with PE anti-NKR-P1A (10/78) andFITC anti-CD3 (1F4).

Figure 11. Flow cytometric analysis of ratsplenocytes stained with PE anti-CD3 (1F4)and FITC anti-CD62L (HRL1).

Figure 8. Flow cytometric analysis of rat peripheral blood monocytes characterized bythe expression of CD4 (OX-35) and lack of CD3 (1F4) (A).The CD3– CD4+ subset wasanalyzed using FITC anti-CD11b (WT.5) (B).

Figure 9. Flow cytometric analysis of rat granulocytes, characterized by a high side scatterprofile (A). Gated granulocytes were stained with APC anti-granulocyte marker (HIS48) (B).

Figure 10. Immunohistochemical staining of frozen rat spleen with anti-CD29(Ha2/5) (A) and flow cytometric analysis of rat splenocytes stained with PE anti-CD3 (1F4) and FITC anti-CD29 (Ha2/4) (B).

NK Cells

Granulocytes

Adhesion Molecules

Monocytes

Dog Immunophenotyping


A.

PE anti-B cells (LSM11.425)100 101 102 103 104

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Figure 14. Activation-induced changes inthe level of expression of canine activationmarker (CTL2.58) on peripheral bloodlymphocytes following in vitro incubationfor 72 hours with media alone (openhistogram) or with Concanavalin A(shaded histogram).

Figure 12. Canine peripheral bloodlymphocytes stained with anti-canine B cells purified antibody (LSM11.425),followed by PE Rat anti-mouse IgG1. Fordata analysis, a gate was set to includeonly viable lymphocytes.

Figure 13. Canine peripheral blood lymphocytes stained with anti-canine CD8 purified antibody (LSM1.140) (A), anti-CD4purified antibody (LSM12.125) (B), and anti-Pan T cell purified antibody (LSM8.853) (C), followed by FITC Rat anti-mouseIgG1. For data analysis, a gate was set to include viable cells.*

Figure 15. Canine peripheral blood. Theshaded histogram represents stainingwith purified anti-CD44 (69.55) and theopen histogram represents the isotypecontrol. FITC-conjugated goat anti-mouseIg was used as a second-step reagent.

B lymphocytes

T lymphocytes

* The reagents used in this experiment are coming soon.

Baboon Rhesus Cynomolgus

Non-Human Primate Immunophenotyping



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Figure 18. Flow cytometric analysis of CD3 expression on peripheral blood lymphocytes of non-human primates.

Figure 17. Flow cytometric analysis of CD20 expression on peripheral blood leukocytes of non-human primates.

Figure 16. Flow cytometric analysis of CD45 expression on peripheral blood leukocytes ofnon-human primates.

T lymphocytes

B lymphocytes

PerCP anti-CD3 (SP34.2) PerCP anti-CD3 (SP34.2)

PE-Cy5 anti-CD45 (Tü116) PE-Cy5 anti-CD45 (Tü116)

PerCP anti-CD3 (SP34.2)

PE anti-CD20 (2H7) PE anti-CD20 (2H7) PE anti-CD20 (2H7)



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Figure 21. Flow cytometric analysis of CD25 expression on peripheral blood mononuclear cells of non-human primates (thin black line:isotype control, green line: unstimulated PBMCs, red line: Concavalin A-stimulated PBMCs).

T lymphocytes (continued)

PE anti-CD8 (RPA-T8) PE anti-CD8 (RPA-T8) PE anti-CD8 (RPA-T8)

PE anti-CD25 (M-A251) PE anti-CD25 (M-A251) PE anti-CD25 (M-A251)

FITC anti-CD4 (M-T477) FITC anti-CD4 (M-T477) FITC anti-CD4 (M-T477)

Non-Human Primate Immunophenotyping (continued)




100 101 102 103 104100

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100 101 102 103 104100

101

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100 101 102 103 104100

101

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100 101 102 103 104100

101

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104

Figure 23. Flow cytometric analysis of CD69 and TNF expression in peripheral blood mononuclear cells of non-human primates.

Figure 24. Flow cytometric analysis of CD69 and IFN-γ expression in stimulated peripheral blood lymphocytes of non-human primates.

Baboon Rhesus

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Figure 22. Flow cytometric analysis of CD11b expression on peripheral blood granulocytesof non-human primates.

Granulocytes

Cytokines

Purified anti-CD11b (ICRF44) Purified anti-CD11b (ICRF44)

FITC anti-TNF (Mab11)

PE a

nti

-CD

69 (

FN50

)

PE a

nti

-CD

69 (

FN50

)

PE a

nti

-CD

69 (

FN50

)

PE a

nti

-CD

69 (

FN50

)

PE a

nti

-CD

69 (

FN50

)

PE a

nti

-CD

69 (

FN50

)

FITC anti-TNF (Mab11) FITC anti-TNF (Mab11)

FITC anti-IFN-γ (4S.B3) FITC anti-IFN-γ (4S.B3) FITC anti-IFN-γ (4S.B3)

Flow cytometry is an analytical tool that allows for the discrimination of different particleson the basis of size and color. The BD™ CBA employs a series of particles with discretefluorescence intensities to simultaneously detect multiple soluble analytes from a singleserum, plasma, or tissue culture supernatant sample. The BD CBA, combined with flowcytometry, creates a powerful multiple analyte (multiplex) assay system.

The BD CBA system uses the sensitivity of amplified fluorescence detection by flow cytometryto measure soluble analytes in a particle-based immunoassay. The combined advantagesof the broad dynamic range of fluorescence detection via flow cytometry and the efficientcapturing of analytes via suspended particles coated with distinct capture antibodies enablethe BD CBA to use fewer sample dilutions to determine analyte concentration in substantiallyless time (compared to conventional ELISA).

The specific capture beads are mixed with the phycoerythrin-conjugated detection antibodiesand then incubated with recombinant protein standards or test samples to form sandwichcomplexes. Following acquisition of sample data using the flow cytometer, the sample resultsare generated in graphical and tabular format using the BD CBA Software.

Typical BD CBA Assay Protocol

1. Add unknown(s) or standards to capture bead array.

2. Add detection reagents and incubate.

3. Wash and acquire samples.

4. Perform batch analysis using BD CBA Software.

Note: Single analyte shown. Always refer to kitprotocol for specific instructions.


+

or

+

W A S H

I N C U B A T E

A N A L Y Z E

A C Q U I R E

BD CBA

BD™ Cytometric Bead Array (BD CBA) System

Figure 25. Representative Non-Human PrimateReactivity. Cynomolgus macaque serum tested usingthe BD CBA Human Inflammation Kit.

Many BD CBA customers have determined that they are able to detect positive signals for rhesus and cynomolgus macaque sampleswhen using the BD CBA Human Th1/Th2 Cytokine Kit. The BD CBA results have been confirmed by ELISA using the BD CBA antibodypairs with activated cell culture samples from both rhesus and cynomolgus macaques. The cross-reactivity of BD CBA Human assayswith non-human primate (NHP) analytes have not yet been normalized to native or recombinant NHP proteins, so direct quantitation is not yet available (Table 2, Figure 25).

Non-Human Primate (continued)



100 101 102 103 104100

101

102

103

104

Measured Cytokine (PE)

FL3-

Hei

gh

t

Interleukin-8

Interleukin-6

Human Chemokine Kit I CXCL8/IL-8, CCL5/RANTES, CCL2/MCP-1 552990

Human Th1/Th2 Cytokine Kit Interleukin (IL)-4, IL-5, TNF, IFN-γ 550749

Human Th1/Th2 Cytokine Kit II Interleukin (IL)-4, IL-6, TNF, IFN-γ 551809

Human Inflammation Kit I Interleukin (IL)-8, IL-6, TNF 551811

(IL-1β and IL-12p70 not yet tested)

BD Cytometric Bead Array (BD CBA) Kits

Table 2. BD CBA Cross-Reactivity with Non-Human Primate Cytokines and Chemokines

DESCRIPTION RHESUS AND CYNOMOLGUS CROSS-REACTIVITY CAT. NO.

BD Cytometric Bead Array System

BD BrdU Flow Kit FCM 50 tests 559619

BD BrdU APC Flow Kit FCM 50 tests 552598 ■

NEW

Rat µicroflow Basic Kit Rat FCM 60 tests 552729

Rat µicroflow Plus Kit Rat FCM 60 tests 552731

Mouse µicroflow Basic Kit Ms FCM 60 tests 552728

Mouse µicroflow Plus Kit Ms FCM 60 tests 552730

Micronucleus Analysis Assay Reagents

DESCRIPTION REACT APPS SIZE CAT. NO.

BrdU Assay Reagents

CD2 (LFA-2) Rat OX-34 Mouse IgG2a, κ FCM, IHC(F), IHC(Fr), IP Purified 0.5 mg 554826

FCM FITC 0.5 mg 554827

CD3 Rat 1F4 Mouse (BALB/c) IgM, κ Cyt, FCM, IP Purified 0.5 mg 556970


FCM PE 0.1 mg 550353

FCM APC 0.1 mg 557030

G4.18 Mouse (BALB/c) IgG3, κ Block, Cost, Cyt, FCM NA/LE 0.5 mg 554829

Cost, Cyt, FCM, IP Purified 0.1 mg 559974

Cost, Cyt, FCM, IP Purified 0.5 mg 554830

IHC(F), IHC(Fr), IHC(Zn) Purified 1 ml 550295

FCM Biotin 0.5 mg 554831



FCM PE 0.2 mg 554833

G4.18/G1 Mouse (BALB/c) IgG1, κ Cost, Cyt, FCM, IP Purified 0.5 mg inquire ■

FCM PE 0.2 mg inquire ■

CD4 Rat OX-35 Mouse (BALB/c) IgG2a, κ FCM, IP Purified 0.5 mg 554835

IHC(Fr) Purified 1 ml 550296



FCM PE 0.2 mg 554838

FCM PE-Cy5 0.1 mg 554839

FCM APC 0.1 mg 550057

OX-38 Mouse (BALB/c) IgG2a, κ FCM, IP Purified 0.5 mg 554841



FCM PE 0.1 mg 551397

CD5 Rat OX-19 Mouse IgG1, κ FCM, IHC(Fr), IHC(Zn), IP Purified 0.5 mg 554849

FCM FITC 0.1 mg 551449 ■

FCM PE 0.2 mg 554851

Rat ReagentsAntibodies to Rat Leukocytes and Related Cells

DESCRIPTION REACT CLONE ISOTYPE APPS FORMAT SIZE CAT. NO.


NEW

DESCRIPTION APPS SIZE CAT. NO.NEW

Reagents for Immunotoxicology Research

CD6 Rat OX-52 Mouse (BALB/c) IgG2a, κ FCM, IHC(Fr), IP Purified 0.1 mg 550979


CD8a Rat G28 Mouse (BALB/c) IgG2a, κ FCM, IP Purified 0.1 mg 559977


OX-8 Mouse (BALB/c) IgG1, κ FCM, IP, WB Purified 0.5 mg 554854


FCM, WB Biotin 0.5 mg 554855


FCM PE 0.1 mg 559976

FCM PE 0.2 mg 554857

FCM PerCP 0.1 mg 558824

CD8b Rat 341 Mouse (BALB/c) IgG1, κ FCM, IHC(Fr), IHC(Zn), Purified 0.5 mg 554971IP, WB

FCM Biotin 0.1 mg 550970 ■


CD9 Rat RPM.7 Mouse IgG3, κ FCM, IP, WB Purified 0.1 mg 551808


CD11a (Integrin αL chain, Rat WT.1 Mouse (BALB/c) IgG2a, κ FCM, IHC(Fr), IP Purified 0.1 mg 559979LFA-1 α chain)


FCM PE 0.1 mg 550972

CD11b (Integrin αM chain, Mac-1 α chain) Rat WT.5 Mouse (BALB/c) IgA, κ FCM, IHC(Fr), IP, WB Purified 0.5 mg 554980



CD11b/c Rat OX-42 Mouse (BALB/c) IgG2a, κ Block, FCM NA/LE 0.5 mg 554858

FCM, IP Purified 0.5 mg 554859

IHC(Fr), IHC(Zn) Purified 1 ml 550299


FCM PE 0.2 mg 554862

CD18 (Integrin β2 chain) Rat WT.3 Mouse (BALB/c) IgG1, κ Block, FCM NA/LE 0.5 mg 554976

FCM, IHC(Fr), IHC(Zn), IP Purified 0.5 mg 554977


CD24 Rat HIS50 Mouse (BALB/c) IgM, κ FCM, IF, IHC(Fr), WB Purified 0.1 mg 551133

CD25 (IL-2R α chain) Rat OX-39 Mouse (BALB/c) IgG1, κ FCM, IHC(Fr), IP Purified 0.1 mg 559980



FCM PE 0.2 mg 554866

CD26 Rat OX-61 Mouse (BALB/c) IgG2a, κ FCM, IHC(Fr), IP, WB Purified 0.1 mg 559639

FCM PE 0.1 mg 559641

CD27 Ms, Rat LG.3A10 Armenian Hamster IgG1, κ Cost, FCM NA/LE 0.5 mg 553777


FCM, IC/FCM PE 0.1 mg 558754

CD28 Rat JJ316 Mouse (BALB/c) IgG1, κ Cost, FCM, IP NA/LE 0.5 mg 554992

JJ319 Mouse (BALB/c) IgG1, κ Cost, FCM NA/LE 0.5 mg 554993

Cost, FCM, IP Purified 0.1 mg 559982


FCM PE 0.1 mg 559984

Rat Reagents (continued)

Antibodies to Rat Leukocytes and Related Cells




Reagents for Immunotoxicology Research (continued)

NEW


CD29 (Integrin β1 chain) Ms, Rat Ha2/5 Armenian Hamster IgM, κ Block, FCM NA/LE 0.5 mg 555002




HMβ1-1 Armenian Hamster IgG2, λ Block, FCM, IP Purified 0.5 mg 553837


CD31 (PECAM-1) Pig, Rat TLD-3A12 Mouse (BALB/c) IgG1, κ Block, FCM NA/LE 0.5 mg 555024

ELISA, FCM, IP Purified 0.5 mg 555025



FCM PE 0.1 mg 555027

CD32 (FcγII Receptor) Rat D34-485 Mouse (BALB/c) IgG1, κ Block, FCM NA/LE 0.5 mg 550273

Block, FCM, IHC(Fr), Purified 0.1 mg 550270IHC(Zn), WB

Block, FCM, IHC(Fr), Purified 0.5 mg 550271IHC(Zn), WB


CD36 (Scavenger Receptor) Rat CRF D-2712 Mouse IgA, κ FCM, ICC Purified 0.1 mg 552554

CD40 Ms, Rat HM40-3 Armenian Hamster IgM, κ Block, Cost, FCM NA/LE 0.5 mg 553721

FCM Purified 0.1 mg 553722


CD42d (Platelet Glycoprotein V) Rat RPM.4 Mouse (BALB/c) IgG2a, κ FCM, IP Purified 0.5 mg 557239


Ms, Rat 1C2 Armenian Hamster IgG3, κ FCM, IP, IHC (Fr) Purified .05 mg 552992

CD43 (Leukosialin) Rat HIS17 Mouse (BALB/c) IgG1, κ FCM, IHC(F), IHC(Fr), Purified 0.5 mg 554867IHC(Zn)


CD44H (Pgp-1, H-CAM) Rat OX-49 Mouse (BALB/c) IgG2a, κ FCM, IHC(F), IHC(Fr), Purified 0.5 mg 554869IHC(Zn), IP


CD45 (Leukocyte Common Antigen) Rat OX-1 Mouse (BALB/c) IgG1, κ FCM, IP Purified 0.5 mg 554875

IHC(F)*, IHC(Fr), Purified 1 ml 550566IHC(Zn)



FCM PE 0.2 mg 554878

FCM PE-Cy5 0.1 mg 559135

CD45.1 (RT7.1 of LCA) Rat NDS58 Rat (AS) IgG2b, κ FCM FITC 0.1 mg 550802

CD45.2 (RT7.2 of LCA) Rat HIS41 Mouse (BALB/c) IgG1, κ FCM, IHC(Fr) Purified 0.1 mg 559985


CD45R Rat HIS24 Mouse (BALB/c) IgG2b, κ FCM, WB Purified 0.5 mg 554879



FCM PE 0.2 mg 554881

CD45RA Rat OX-33 Mouse (BALB/c) IgG1, κ FCM Purified 0.5 mg 554882



FCM PE 0.1 mg 551402

FCM PE 0.2 mg 554884

FCM PE-Cy5 0.1 mg 557015

CD45RC Rat OX-22 Mouse (BALB/c) IgG1, κ FCM Purified 0.1 mg 551451 ■


FCM PE 0.2 mg 554888



DESCRIPTION REACT CLONE ISOTYPE APPS FORMAT SIZE CAT. NO.NEW


CD48 (OX-45 antigen) Rat OX-45 Mouse (BALB/c) IgG1, κ FCM Purified 0.1 mg 552011 ■

CD49a (Integrin α1 chain) Ms, Rat Ha31/8 Armenian Hamster IgG2, λ Block, FCM NA/LE 0.5 mg 555000



CD49b (Integrin α2 chain) Ms, Rat Ha1/29 Armenian Hamster IgG2, λ FCM, IHC(Fr) Purified 0.1 mg 559987

FCM, IHC(Fr) Purified 0.5 mg 554998


CD49c (Integrin α3 chain) Dog, Ms, Rat 42 Mouse IgG1 IF, WB Purified 50 µg 611044

IF, WB Purified 150 µg 611045

CD49d (Integrin α4 chain) Rat MRα4-1 Mouse (BALB/c) IgG2a, κ FCM, IP Purified 0.5 mg 553348


CD49e (Integrin α5 chain) Ms, Rat HMα5-1 Armenian Hamster IgG1, κ FCM, IP Purified 0.5 mg 553350

CD53 Rat OX-44 Mouse (BALB/c) IgG1, κ FCM, IHC(Fr), IHC(Zn), IP Purified 0.1 mg 551452 ■


CD54 (ICAM-1) Rat 1A29 Mouse (BALB/c) IgG1, κ Block, FCM NA/LE 0.5 mg 554966





FCM PE 0.2 mg 554970

CD59 (MAC inhibitor) Rat TH9 Mouse (BALB/c) IgG1, κ FCM FITC 0.1 mg 550976 ■

CD61 (Integrin β3 chain) Ms, Rat 2C9.G2 Armenian Hamster IgG1, κ Block, FCM NA/LE 0.5 mg 553343





FCM PE 0.2 mg 553347

Rat F11 Mouse (BALB/c) IgG1, κ Block, FCM, IP NA/LE 0.5 mg 554950




CD62L (L-selectin, LECAM-1) Rat HRL1 Armenian Hamster IgG2, λ FCM Purified 0.1 mg 559988



FCM PE 0.1 mg 551398

HRL2 Armenian Hamster IgG1, κ ELISA, FCM, IHC(Fr) Purified 0.1 mg 559990

CD62P (P-selectin) Dog, Hu, Ms, Polyclonal Rabbit IgG ELISA, FCM, IHC(Fr), IP, Purified 0.1 mg 553716Rat WB

CD63 (ME491) Rat AD1 Mouse (BALB/c) IgG1, κ FCM, IHC, IP Purified 0.1 mg 551458 ■

CD71 (Transferrin Receptor) Rat OX-26 Mouse (BALB/c) IgG2a, κ FCM, IHC(Fr), IP Purified 0.5 mg 554889


FCM PE 0.2 mg 554891

CD73 (Ecto-5’-nucleotidase) Rat 5F/B9 Mouse (BALB/c) IgG1, κ Cost, FCM NA/LE 0.5 mg 551122

FCM, IF, IHC(Fr), Purified 0.1 mg 551123IHC(Zn), IP

FCM PE 0.1 mg 551124

CD80 (B7-1) Rat 3H5 Mouse (BALB/c) IgG1, κ FCM, IHC(Fr), IP Purified 0.5 mg 555012


FCM PE 0.2 mg 555014






*IHC application not routinely tested by BD Biosciences Pharmingen.


CD81 (TAPA-1) Ms, Rat Eat2 Armenian Hamster IgG1, κ FCM, IP, WB Purified 0.5 mg 559517


FCM PE 0.1 mg 559519

CD86 (B7-2) Rat 24F Mouse (BALB/c) IgG1, κ Block, FCM NA/LE 0.5 mg 555015

FCM, IHC(Fr), IP Purified 0.5 mg 555016



FCM PE 0.1 mg 551396

CD90.1 (Thy-1.1) Ms, Rat HIS51 Mouse (BALB/c) IgG2a, κ FCM Purified 0.5 mg 554892




G Pig, Ms, OX-7 Mouse (BALB/c) IgG1, κ Cost, FCM, IP, WB Purified 0.5 mg 554895Rab, Rat




FCM PE 0.1 mg 551401

FCM PE 0.2 mg 554898


CD95 (Fas) Chick, Dog, 13 Mouse IgG1 IF, IHC, WB Purified 50 µg 610197*Hu, Ms, Rat

CD106 (VCAM-1) Rat MR106 Mouse IgG1, κ FCM, IHC(Fr), IP, WB Purified 0.5 mg 559165

FCM PE 0.1 mg 559229

CD122 (IL-2 Receptor β chain) Rat L316 Mouse (BALB/c) IgG1, κ FCM Biotin 0.5 mg 557282


CD134 (OX-40 Antigen) Rat OX-40 Mouse (BALB/c) IgG2b, κ FCM Biotin 0.1 mg 550977


CD140b (PDGF Receptor β chain) Chick, Dog, 28 Mouse IgG2b IF, IHC, IP, WB Purified 50 µg 610113*Hu, Ms, Rat

IF, IHC, IP, WB Purified 150 µg 610114*

CD147 Rat OX-47 Mouse IgG1, κ FCM Purified 0.1 mg 552012 ■

CD161a (NKR-P1A) Rat 10/78 Mouse IgG1, κ FCM, IP Purified 0.5 mg 555006




FCM PE 0.2 mg 555009

CD172 (SIRP) Rat OX-41 Mouse IgG2a,κ FCM, IHC, IP, WB Purified 0.1 mg 552297 ■

FCM PE 0.1 mg 552298 ■

CD178 (Fas Ligand, CD95 Ligand) Dog, Hu, Ms, 33 Mouse IgG1 IF, IP, WB Purified 50 µg 610410Rat

IF, IP, WB Purified 150 µg 610411

Ms, Rat MFL4 Armenian Hamster IgG3, κ Block, FCM NA/LE 0.5 mg 555021



CD200 (OX-2 Antigen) Rat OX-2 Mouse IgG1, κ Block, FCM, IHC, IP Purified 0.1 mg 552457 ■

CD200 (OX-2 Receptor) Rat OX-102 Mouse (BALB/c) IgG1, κ FCM, IP, WB Purified 0.1 mg 552469 ■

β2 Microglobulin Rat TLD-3H12B Mouse (BALB/c) IgG1, κ FCM, IHC, WB Purified 0.5 mg 558765

FCM FITC 0.1 mg 551807 ■





C1qRp Rat LOV8 Mouse IgG1, λ FCM, IP Purified 0.1 mg 552294 ■

FCM PE 0.1 mg 552295 ■

Crry/p65 Rat 512 Mouse (BALB/c) IgG1, κ FCM, WB Purified 0.5 mg 554991

Dendritic Cells Rat OX-62 Mouse (BALB/c) IgG1, κ FCM, IP, WB Purified 0.5 mg 555010


Erythroid Cells Rat HIS49 Mouse (BALB/c) IgM, κ FCM, IHC(Fr) Purified 0.1 mg 550961


Granulocytes Rat HIS48 Mouse IgM, κ FCM Purified 0.5 mg 554905




RP-1 Mouse (BALB/c) IgG2a, κ FCM, IP Purified 0.5 mg 550000


FCM PE 0.1 mg 550002

RP-3 Mouse (BALB/c) IgM, κ Cyt, FCM NA/LE 0.5 mg 550055


High Affinity IgE Receptor (FcεRI) Rat BC4 Mouse (BALB/c) IgG1, κ FCM, IP Purified 0.1 mg 551469 ■

Ki-67 Hu, Ms, Pig, B56 Mouse IgG1, κ IC/FCM FITC Set 100 tests 556026Rat

IC/FCM PE Set 100 tests 556027

Ly49 Inhibitory Receptor 2 Rat STOK2 Rat (DA) IgG2a, κ Block, FCM, IP Purified 0.1 mg 552296 ■

Macrophage Activator (RMA) Rat anti-RMA Mouse IgG1, κ FCM, IHC(Fr), IP Purified 0.5 mg 555020

Macrophage Subset Rat HIS36 Mouse IgG2a, κ FCM Purified 0.5 mg 554900


FCM PE 0.2 mg 554901

MAdCAM-1 Rat OST2 Mouse IgG1, κ FCM, IP Purified 0.1 mg 550014


Marginal Zone B Cells Rat HIS57 Mouse IgG1, κ FCM, IHC(F), IHC(Fr) Purified 0.1 mg 559960



Mast Cells Rat AR32AA4 (aka Mouse (BALB/c) IgG1, κ FCM Purified 0.1 mg 551770 ■

AA4)

Mononuclear Phagocyte Rat 1C7 Mouse IgG1, κ FCM Purified 0.5 mg 554954



Myeloid Lineage Rat OX-82 Mouse (BALB/c) IgG1, κ FCM Purified 0.1 mg 552130 ■

OX-40 Ligand (OX-40L) Rat ATM-2 Mouse (BALB/c) IgG1, κ FCM Purified 0.5 mg 559957


RT6.1 Rat P4/16 Rat IgG2b κ FCM, WB Purified 0.1 mg 552725 ■

RT6.2 Rat GY1/12 Rat IgG2c, κ FCM, WB Purified 0.1 mg 552471 ■






RT1A Rat OX-18 Mouse (BALB/c) IgG1, κ FCM, IHC(Fr), IP Purified 0.5 mg 554917

FCM, IHC(Fr) Biotin 0.1 mg 550980


FCM PE 0.1 mg 559993

RT1Aa,b Rat C3 Rat (LOU/cN) IgG2b, κ FCM Purified 0.5 mg 554932



RT1Aa,b,l Rat B5 Rat (LOU/cN) IgM, κ FCM Purified 0.1 mg 559995


RT1B Ms, Rat OX-6 Mouse (BALB/c) IgG1, κ FCM, IP Purified 0.5 mg 554926

IHC(F), IHC(F)*, Purified 1 ml 550574IHC(Fr), IHC(Zn)



FCM PE 0.2 mg 554929


RT1D Ms, Rat 14-4-4S Mouse (C3H.SW) IgG2a, κ Cyt, FCM, IHC(Fr), IP Purified 0.1 mg 558734



FCM PE 0.1 mg 553544

RT1D Rat OX-17 Mouse (BALB/c) IgG1, κ FCM FITC 0.1 mg 550982

Antibodies to Rat Major Histocompatibility Complex (MHC) Antigens


αβ TCR Rat R73 Mouse (BALB/c) IgG1, κ Cost, Cyt, FCM NA/LE 0.5 mg 554910

Cost, Cyt, FCM, IP, WB Purified 0.5 mg 554911


FCM Biotin 0.1 mg 551801 ■


FCM PE 0.2 mg 554914


γδTCR Rat V65 Mouse (BALB/c) IgG1, κ Cost, Cyt, FCM, IF, IP Purified 0.5 mg 554956




FCM PE 0.1 mg 551802

Vα 4 TCR Rat G99 Mouse IgG1, κ Cost, FCM, IHC(Fr) Purified 0.5 mg 554989

Vβ 3.3 TCR Rat C-A11 Mouse (BALB/c) IgG2a, κ FCM Biotin 0.1 mg 550731

Vβ 8.2l and Vβ 8.4a TCR Rat R78 Mouse (BALB/c) IgG1, κ Cost, Cyt, FCM, IHC(Fr) Purified 0.5 mg 554940


Vβ 13 TCR Rat 18B1 Mouse (BALB/c) IgG1, κ FCM Biotin 0.1 mg 550721

Vβ 16 TCR Rat HIS42 Mouse (BALB/c) IgG2b, κ Cost, FCM, IHC(Fr) Purified 0.5 mg 554915

Antibodies to Rat T-Cell Receptors (TCR)



NEW

NEW

Rat IFN-γ Set 31.3-2000 pg/ml ELISA Reagents for 20 Plates 558861

Rat IL-2 Set 31.3-2000 pg/ml ELISA Reagents for 20 Plates 557044


Rat IL-6 Set 78-5000 pg/ml ELISA Reagents for 20 Plates 550319


Rat MCP-1 Set 31.3-2000 pg/ml ELISA Reagents for 20 Plates 555130

Rat TNF Set 31.3-2000 pg/ml ELISA Reagents for 20 Plates 558870

BD OptEIA™ ELISA Sets

DESCRIPTION ASSAY RANGE APPS SIZE CAT. NO.

Rat TNF Kit 31.3-2000 pg/ml 13 pg/ml ELISA 2 plates 550734

BD OptEIA™ ELISA Kits

DESCRIPTION ASSAY RANGE SENSITIVITY APPS SIZE CAT. NO.

CD11a (LFA-1) Dog, Hu, Rab HI111 Mouse IgG1, κ FA, FCM, IHC(Fr) NA/LE 0.5 mg 555381



FCM FITC 100 tests 555383

FCM PE 100 tests 555384


FCM PE-Cy5 100 tests 551131 ■

FCM APC 100 tests 559875


CD14 Dog, Hu M5E2 Mouse IgG2a, κ FCM NA/LE 0.5 mg 555395









FCM PerCP-Cy5.5 0.1 mg 550787

CD34 Dog 1H6 Mouse (BALB/c) IgG1, κ FCM PE 0.1 mg 559369

2E9 Mouse (BALB/c) IgG1, κ FCM Biotin 0.1 mg 550427

DogAntibodies to Dog Leukocytes





NEW

NEW

NEW


CD44 (Pgp-1, Ly-24) Dog 69-S5 Mouse (BALB/c) IgG1, κ FCM, IF Purified 0.1 mg 559964

Dog, Hu, Ms, Pig IM7 Rat IgG2b, κ Block, Cyt, FCM NA/LE 0.5 mg 553130

Cyt, ELISA, FCM, IP Purified 0.5 mg 553131




FCM PE 0.1 mg 553134

FCM PE-Cy5 0.1 mg 553135

FCM APC 0.1 mg 559250

CD49c (Integrin α3 chain) Dog, Ms, Rat 42 Mouse IgG1 IF, WB Purified 50 µg 611044

IF, WB Purified 150 µg 611045

CD49d (Integrin α4 chain) Dog, Hu 9F10 Mouse IgG1, κ FCM NA/LE 0.5 mg 555501




FCM PE-Cy5 100 tests 559880


CD49f (Integrin α6 chain) Dog, Hu, Ms, Pig GoH3 Rat (Sprague-Dawley) FCM, IHC(Fr), IP Purified 0.1 mg 555734IgG2a, κ




FCM APC 100 tests 551140 ■

CD58 (LFA-3) Dog, Hu 1C3 Mouse IgG2a, κ FCM Purified 0.1 mg 555919




CD61 Dog, Hu VI-PL2 Mouse IgG1, κ FCM Purified 0.1 mg 555752





CD62P (P-selectin) Dog, Hu, Ms, Rat Polyclonal Rabbit IgG ELISA, FCM, Purified 0.1 mg 553716IP, WB

CD80 (B7-1) Dog, Ms 16-10A1 Armenian Hamster IgG2, κ FCM, IHC(Fr), IP Purified 0.5 mg 553766



FCM PE 0.2 mg 553769

Dog, Ms 1G10 Rat IgG2a, κ FCM Purified 0.5 mg 553368

CD81 Dog, Hu, Rab JS-81 Mouse IgG1, κ FCM Purified 0.1 mg 555675





CD94 Dog, Hu HP-3D9 Mouse IgG1, κ FCM, IHC(Fr) Purified 0.1 mg 555887




Dog (continued)

Antibodies to Dog Leukocytes



*IHC application not routinely tested by BD Biosciences Pharmingen.

CD95 (Fas/APO-1) Dog, Hu, Pig DX2 Mouse IgG1, κ FA, FCM NA/LE 0.5 mg 555670


FCM Biotin 100 tests 555672





CD117 (c-kit) Dog, Hu, Rab YB5.B8 Mouse IgG1, κ FCM Purified 0.1 mg 555713

FCM PE 0.1 mg 555714

FCM PE-Cy5 0.1 mg 559879

FCM APC 0.1 mg 550412

CD132 (common γ chain) Dog, Hu TUGh4 Rat IgG2b, κ FA, FCM NA/LE 0.5 mg 555895



FCM PE 0.2 mg 555898

CD140b (PDGF Receptor β chain) Chick, Dog, Hu, 28 Mouse IgG2b IF, IHC, IP, WB Purified 50 µg 610113*Ms, Rat

IF, IHC, IP, WB Purified 150 µg 610114*

CD178 (Fas Ligand, CD95 Dog, Hu, Ms, Rat 33 Mouse IgG1 IF, IP, WB Purified 50 µg 610410Ligand)

IF, IP, WB Purified 150 µg 610411

HLA-DR Dog, Hu G46-6 Mouse IgG2a, κ FCM NA/LE 0.5 mg 555809









Dog, Hu, Rab TÜ36 Mouse IgG2b, κ FCM Purified 0.1 mg 555559





SLA-DR Bov, Cat, Dog, 1F12 Mouse (BALB/c) IgG2b, κ FCM, IP, WB Purified 0.1 mg 551537 ■

G Pig, Gt,Hs, Hu,Mink, Pig, Sheep

VAP-1 Dog, Ms, Rat 54 Mouse IgG1 WB Purified 50 µg 611190

WB Purified 150 µg 611191

Dog (continued)

Antibodies to Dog Leukocytes





CCR6 Cyno, Rhe 11A9 Mouse IgG2a, κ FCM PE 50 tests 551773 ■

CD2 Bab, Cyno, Rhe RPA-2.10 Mouse IgG1,κ FCM, IHC(Fr) Purified 0.1 mg 556607



CD3 Bab, Cyno, Rhe SP34 Mouse IgG3, λ FA, FCM NA/LE 0.5 mg 557052

FA, FCM Purified 0.1 mg 556610

FA, FCM FITC 50 tests 556611

FA, FCM PE 50 tests 556612

SP34-2 Mouse IgG1, κ FCM Purified 0.1 mg 551916 ■

FCM PE 50 tests 552127 ■

CD3/CD16/CD45 Rhe SP34, 3G8, TÜ116 FCM FITC, PE, PerCP 50 tests 551067

CD3/CD20/CD45 Rhe SP34, 2H7, TÜ116 FCM FITC, PE, PerCP 50 tests 551068

CD3/CD4/CD45 Rhe SP34, M-T477, TÜ116 FCM FITC, PE, PerCP 50 tests 551070

CD3/CD8/CD45 Rhe SP34, RPA-T8, TÜ116 FCM FITC, PE, PerCP 50 tests 551069

CD4 Bab, Cyno, Rhe L200 Mouse IgG1, κ FCM Purified 0.1 mg 550625



FCM PerCP 50 tests 550631

M-T477 Mouse IgG2a FCM Purified 0.1 mg 556614



CD4/CCR5 Cyno, Rhe M-T477, 3A9 FCM FITC, PE 50 tests 551466 ■

CD4/CXCR4 Bab, Cyno, Rhe M-T477, 12G5 FCM FITC, PE 50 tests 551465 ■

CD6 Bab, Cyno, Rhe M-T605 Mouse IgG1, κ FCM FITC 50 tests 559491


CD8 Bab, Cyno, Rhe RPA-T8 Mouse IgG1, κ FCM Purified 0.1 mg 557084


CD8/CCR5 Bab, Cyno, Rhe RPA-T8, 3A9 FCM FITC, PE 50 tests 551470 ■

CD8/CXCR4 Bab, Cyno, Rhe RPA-T8, 12G5 FCM FITC, PE 50 tests 551468 ■

CD9 Bab, Cyno, Rhe M-L13 Mouse IgG1, κ FCM, IHC(Fr), WB Purified 0.1 mg 559456


CD10 Bab, Cyno, Rhe HI10a Mouse IgG1, κ FCM PE 50 tests 557143

CD11a/LFA-1 Bab, Cyno, Rhe HI111 Mouse IgG1, κ FCM PE 50 tests 550851


CD11b/Mac-1 (CR3) Bab, Cyno, Rhe ICRF44 Mouse IgG1, κ FCM Purified 0.1 mg 557320


CD14 Cyno, Rhe M5E2 Mouse IgG2a, κ FCM Purified 0.1 mg 557152



CD16 Bab, Cyno, Rhe 3G8 Mouse IgG1, κ FCM Purified 0.1 mg 556617



CD18 (Integrin β2 chain, Cyno, Rhe 6.7 Mouse IgG1, κ FCM FITC 50 tests 557156CR3/CR4)


CD20 Bab, Cyno, Rhe 2H7 Mouse IgG2b, κ FCM Purified 0.1 mg 556631



CD21 Bab, Cyno, Rhe B-ly4 Mouse IgG1, κ FCM PE 50 tests 557327

CD25 Bab, Cyno, Rhe M-A251 Mouse IgG1, κ FCM PE 50 tests 557138

Non-Human Primate (NHP)Antibodies for Non-Human Primate (NHP) Research



CD27 Bab, Cyno, Rhe M-T271 Mouse IgG1, κ FCM Purified 0.1 mg 557328



CD28 Bab, Cyno, Rhe CD28.2 Mouse IgG1, κ FCM Purified 0.1 mg 556620



CD29 Bab, Rhe MAR4 Mouse IgG1, κ FCM Purified 0.1 mg 557331


CD31 Bab, Cyno, Rhe WM59 Mouse IgG1, κ FCM FITC 50 tests 557508

CD32 Bab, Rhe FLI8.26 (2003) Mouse IgG2b, κ FCM Purified 0.1 mg 557333


CD34 Cyno, Rhe 563 Mouse IgG1, κ FCM PE 50 tests 550619

CD40 Bab, Cyno, Rhe 5C3 Mouse IgG1, κ FCM Purified 0.1 mg 556623



CD41a Bab, Cyno, Rhe HIP8 Mouse IgG1, κ FCM Purified 0.1 mg 557295



CD41b Bab, Cyno, Rhe HIP2 Mouse IgG3, κ FCM Purified 0.1 mg 557293


CD42a Bab, Cyno, Rhe ALMA.16 Mouse IgG1, κ FCM Purified 0.1 mg 559458


CD45 Cyno, Rhe TÜ116 Mouse IgG1, κ FCM PE 50 tests 557059


FCM PerCP 50 tests 557513

FCM PerCP-Cy5.5 50 tests 552724

CD45RA Bab, Cyno, Rhe 5H9 Mouse IgG1, κ FCM Purified 0.1 mg 556625



CD49b Bab, Cyno, Rhe AK-7 Mouse IgG1, κ FCM FITC 50 tests 557338

CD49d (Integrin α4 chain) Bab, Cyno, Rhe 9F10 Mouse IgG1, κ FCM Purified 0.1 mg 556634


CD49f Bab, Cyno, Rhe GoH3 Mouse IgG1, κ FCM FITC 50 tests 557510


CD56 Bab, Cyno, Rhe MY31 Mouse IgG1, κ FCM PE 50 tests 556647

CD59 Bab, Cyno, Rhe p282 (H19) Mouse IgG2a, κ FCM PE 50 tests 557141 ■

CD61 Bab, Cyno, Rhe VI-PL2 Mouse IgG1, κ FCM FITC 50 tests 557291

CD62L Cyno, Rhe SK11 Mouse IgG2a, κ FCM PE 50 tests 557341 ■

CD62P (P-Selectin) Bab, Cyno, Rhe AC1.2 Mouse IgG1 FCM FITC 50 tests 550866


CD63 Bab, Cyno, Rhe H5C6 Mouse IgG1, κ FCM Purified 0.1 mg 557304


CD69 Bab, Cyno, Rhe FN50 Mouse IgG1, κ FCM FITC 50 tests 557049


CD83 Cyno, Rhe HB15e Mouse IgG1, κ FCM PE 50 tests 550634

CD86 Bab, Cyno, Rhe 2331 (FUN-1) Mouse IgG1, κ FCM FITC 50 tests 557343


CD95 Bab, Cyno, Rhe DX2 Mouse IgG1, κ FCM Purified 0.1 mg 556639



Non-Human Primate (NHP) (continued)

Antibodies for Non-Human Primate (NHP) Research





CD122 Bab, Cyno, Rhe Mikβ2 Mouse IgG2a, κ FCM Purified 0.1 mg 557322

FCM PE 0.1 mg 557323

CD130 Bab, Cyno, Rhe AM64 Mouse IgG1, κ FCM Purified 0.1 mg 557324


CDw137 Bab, Cyno, Rhe 4B4-1 Mouse IgG1, κ FCM PE 50 tests 557303

CD152 Bab, Cyno, Rhe BNI3 Mouse IgG2a, κ FCM Purified 0.1 mg 557300


CD154 Bab, Cyno, Rhe TRAP1 Mouse IgG1, κ FCM Purified 0.1 mg 557298



CD162 Bab, Cyno, Rhe KPL-1 Mouse IgG1, κ FCM Purified 0.1 mg 557502


CD183 (CXCR3) Cyno, Rhe 1C6/CXCR3 Mouse IgG1 FCM PE 50 tests 550633

CD184 (CXCR4, Fusin) Bab, Cyno, Rhe 12G5 Mouse IgG2a, κ FCM PE 50 tests 557145

CD195 (CCR5) Cyno, Rhe 3A9 Mouse IgG2a FCM PE 50 tests 550632

HLA-A,B,C Bab, Cyno, Rhe G46-2.6 Mouse IgG1, κ FCM Purified 0.1 mg 557347



HLA-DR Bab, Cyno, Rhe G46-6 Mouse IgG2a, κ FCM Purified 0.1 mg 556642



FCM PerCP-Cy5.5 50 tests 552764 ■

HLA-DR,P,Q Bab, Cyno, Rhe TÜ39 Mouse IgG2a, κ FCM FITC 50 tests 550853

IFN-γ Bab, Cyno, Rhe 4S.B3 Mouse IgG1, κ IC/FCM PE 50 tests 557074

IC/FCM APC 50 tests 551385 ■

IL-2 Bab, Cyno, Rhe MQ1-17H12 Rat IgG2a IC/FCM Purified 0.1 mg 558881

IC/FCM FITC 50 tests 559361

IC/FCM APC 50 tests 551383 ■

IL-16 Bab, Cyno, Rhe 14.1 Mouse IgG2a, κ IC/FCM PE 50 tests 551471 ■

APC 50 tests 551472 ■

MCP-1 (monocyte Cyno, Rhe 5D3-F7 Mouse IgG1, κ FCM PE 50 tests 557066 ■

chemoattractant protein 1)


MIP-1α (macrophage Cyno, Rhe 11A3 Mouse IgG2a,κ FCM APC 50 tests 551533 ■

inflammatory protein-1α)

Pan Leukocyte DO58-1283 Mouse IgG1 FCM Purified 0.1 mg 552566 ■

Red Blood Cells (RBC) Bab E34-1678 Mouse IgM FCM Purified 0.1 mg 551350 ■

FCM FITC 50 tests 551351 ■

E34-731 Mouse IgG1, κ FCM Purified 0.1 mg 551299 ■

FCM FITC 50 tests 551300 ■

FCM PE 50 tests 551301 ■

TNF Bab, Cyno, Rhe Mab11 Mouse IgG1, κ IC/FCM Purified 0.1 mg 558882

IC/FCM PE 50 tests 557068

Non-Human Primate (NHP) (continued)

Antibodies for Non-Human Primate (NHP) Research


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