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In vivo

• experiments conducted inside living organism

• Essential for pre-clinical research before human trials.

• These studies are vital in biomedical research because they provide insights into the safety, efficacy, and biological effects of new drugs, chemicals, and therapies before testing them in humans.

• White mice

• Abino rabbits

• What are the most common animal models

• Used in pharmacology, toxicology, immunology, genetics, and surgical research.

95-98%

White mice share — genetic similarity with humans

White mice

• Short reproductive cycle, easy to breed.

• Standardized strains ensure reproductibility

• most widely used animal models due to their genetic similarity to humans, ease of breeding, and established role in preclinical studies.

• BALB/c

• Swiss albino mice

Additionally, many standardized strains exist, such as — and, ensuring reproducibility and reliability in experimental results. Their use has led to countless medical breakthroughs, including treatments for diabetes, cancer, and infectious diseases.

Albino rabbits

• Larger body size suitable for surgical and ocular studies.

• Have similar eye structure to humans which is a key in opthamology.

• Similar with white mice, they are used in antibody production and vaccine testing.

• Selection of healthy organisms (6-8 weeks old)

• Acclimatization (temperature, humidity, light)

• Division to control and experimental group

• Drug administration (oral, IV, IP, SC, Topical)

• Observation

• Data collection and analysis

Methodology of white mice and albino rabbit

• 20-26C

• 15-21C

• 12 hours

Housing and handling

• Temperature for mice

• Temperature for rabbits

• Both are typically housed under — hour light/dark cycle

tunnels or cage ladders

For mice, studies show that non-aversive handling methods such as — or — reduce stress and improve welfare compared to tail handling. Individually ventilated cages provide better air quality and lower stress levels.

1. Oral gavage

2. Intraperitoneal

3. SC

4. IV

5. Topical (skin, ocular)

Drug administration techniques for white mice and rabbits

Tail vein injection

IV site for white mice or albino rabbit

• Toxicology testing

• Pharmacokinetics

APPLICATIONS OF WHITE MICE AND ALBINO RABBITS IN PRE-CLINICAL STUDIES

White mice

In preclinical studies, — are essential for systemic and toxicological research. They help evaluate toxicity, test nanomedicine safety, and model wound healing in diabetic conditions.

Albino rabbits

They are widely used in applied biomedical studies. They serve as reliable models for ocular disease and drug delivery testing, while also remaining the gold standard for dermal irritation and cosmetic safety assessments.

White mices

They are advantageous because of their close genetic similarity to humans, rapid breeding, and cost-effective handling, making them the backbone of systemic preclinical research

Albino rabbits

They are valuable due to their eye and skin physiology that mirrors humans, which makes them ideal for ocular and dermal studies. Their larger size also allows more invasive or repeated procedures that aren’t possible in smaller animals.

White mice

Despite their usefulness, white mice have limitations such as results not always translating clinically, restrictions due to their small size, and ethical concerns.

Albino rabbits

They face similar issues, with species differences in drug metabolism, ethical debates over ocular and dermal testing, and higher costs for housing and care compared to mice.

3Rs principle

This emphasizes replacing animals where possible, reducing their numbers, and refining procedures to minimize suffering.

IACUC Institutional animal care and use committee

All animal studies must be approved by — (oversees the specific use of animals by formally reviewing animal use protocols.)

• 3Rs principle

• IACUC

• International guidelines

What are the ethical considerations for white mice and albino rabbits

• In vitro cell culture

• In silico simulation

• Organoids and tissue engineering

Alternative animals to use for white mice or albino rabbits

• In vitro cell culture

• In silico simulation

• Organoids and tissue engineering

ALTERNATIVES

• — systems using human cells allow for detailed cellular-level studies

• — computer simulations predict drug interactions using algorithms.

• — can mimic organ-level responses.

Organoids

— work by mimic the structure and function of real organs from stem cells

True

True or false

• Reproducibility is better with albino rabbits so good with seeing long term effects; White mice lifespan is short

ELISA

• Specific interactions between antigens and antibodies

• Replicating the interaction allows to detect or measure the presence of specific biological molecules.

Pyrogen

___ test in industrial field is often conducted

• Copper, blue

• Clumps (gel clot formation)

• Limulus polyphemus

LAL Test vs. Rabbit Pyrogen test

• Blood of crab has — (— in color), so in med supply testing, blood is applied to the med supply and if — appear, its positive inoenditoxin

• CI name of the organism

Temperature

— is based in rabbit testing

Pyrogen

Horseshoe crab or LAL test is now the gold standard for - testing

Antigens

• Molecular markers, triggers an immune respons

• Found on the surfaces of viruses, bacteria, allergens, parasites, and body cells.

Antibodies

• Immune system proteins

• Recognize and eliminate foreign or harmful substance.

Enzyme immunoassay (EIA)

• Uses enzymes as labels

• Identification and quantification of target molecules.

• The catalytic activity of these enzymes produces a detectable signal, enabling the identification and quantification of target molecules

ELISA

• One component is immobilized onto a solid phase: (1) microtiter plate (2) magnetic particle (3) plastic bead

• Separates bound and unbound reactants

• Enhance sensitivity and specificity

ELISA

• Gold standard among immunoassays

• Safer alternative to Radioimmunoassay

  • Developed in 1970s replacing radioactive isotopes

• Engvall & Perlman

• Van Weemen & Schuurs

Who pioneered ELISA

• independently developed the method by replacing radioactive labels with enzyme conjugates

1. Coating, wash

2. Blocking, wash

3. Detection, wash

4. Final read

ELISA Methodology

Immunoassays

— are laboratory techniques that rely on the specific interaction between antigens and antibodies.

RIA (Radioimmunoassay)

— was considered dangerous mainly because it used radioactive isotopes as labels to detect antigen–antibody reactions.

• Immunoglobulin G

• Human chorionic gonadotropin

ELISA

• Its first applications were measuring — in rabbit serum and detecting — in urine using horseradish peroxidase.

ELISA

It is an enzyme immunoassay that uses an enzyme-labeled antibody or antigen to detect and measure a specific target. The target is bound to a solid phase (e.g., 96-well plate), unbound components are washed away, and a substrate produces a measurable signal.

Direct elisa

Binds antigens, including the desired target, in a simple directly to the plate. An enzyme-conjugated antibody in then added as a probe for the desired analyte.

• horseradish peroxidase (HRP)

• Alkaline phosphatase (AP) with chromogenic substrates.

* Detection systems of ELISA: —

Armoracia rusticana

Horseradishperoxidase is from —

Indirect ELISA

Binds antigens, including the desired target in the sample to the plate. However, it involves two antibodies; a primary and a secondary conjugated antibody.

Sandwich ELISA

The target is bound between a captured antibody (for antigen detection) or capture protein (for antibody detection) and the conjugated detecting antibody

Competitive ELISA

Involves competition between the binding of the sample antigen and conjugated antigen to a specific amount of antibody. The more antigen in the sample, the less conjugated antigen binds and the lower assay signal.

• Pharmacokinetics

• Pharmacodybamis / biomarker analysis

• Immunogenicity

• Drug stability and tissue distribution

• Toxicology / safety biomarkers

APPLICATIONS OF ELISA IN DRUG DEVELOPMENT/PRE-CLINCIAL STUDIES

Horseshoe crabs (Limulus polyphemus)

Source of Limulus Amebocyte Lysate (LAL):

They have blue blood containing amebocytes.

ELISA

It quantifies therapeutic proteins (e.g., monoclonal antibodies, fusion proteins) in animal serum to calculate PK parameters like half-life, Cmax, AUC.

ELISA

— assays quantify proteins indicative of tissue damage, inflammation, or immune response during preclinical safety studies.

Reverse transcription polymerase chain reaction

Converts mRNA to cDNA using reverse transcriptase + PCR amplification

• RNA sequence

• Single stranded DNA (cDNA)

RT PCR

• It is the template for reverse transcriptase

• It is the template for PCR

Reverse transcription polymerase chain reaction

It couples a reverse transcription reaction with PCR-based amplification to generate cDNA from mRNA.

Primers

/— against known coding regions of mRNA will optimize the reaction for a particular transcript of interest, and are also used for cloning. 

Specific primer

This RT PCR primer target a transcript of interest and aid in cloning

Poly-dT

This RT PCR bind to most mRNAs via poly-A tail

False

True or false

• RT PCR is very sensitive and only needs lrage sample amounts

• Two-step reaction

• One-step reaction

RT PCR can be done as:

• — - RT then PCR separately

• — - combined, with specialized enzyme

• Heat RNA to unwind secondary structure

• Reverse transcribe the RNA ot first strand cDNA

• PCR amplify the cDNA

• Analyze the PCR products

RT PCR Methodology

• Denaturation

• Annealing

• Extension

PCR involves repeating cycles of:

• Gel electrophoresis

• Sequencing

• Quantitative

ANALYZE THE PCR PRODUCTS through what methods

poly-A+

Researchers often enrich for — RNA because messenger RNA (mRNA) carries the instructions for making proteins, making it the most informative RNA type for studying gene activity.

Reverse transcription polymerase chain reaction

assess the effect of a drug candidate on gene expression levels, monitor treatment response or evaluate drug toxicity.