Introduction

Serological diagnosis– It is the diagnosis of infectious diseases based upon the  Antigen-Antibody (Ag-Ab) reactions. Ag – Ab reaction is the union of an Ag with its specific Ab, best detected when each is in optimal concentration.  

The type of such reaction depends upon the nature of both Ag and Ab used.  

These reactions are mostly used in the laboratory (In Vitro) to detect either the Ag  (Direct diagnosis) or the Ab (indirect diagnosis).

FEATURES AND KINETICS OF THE  HUMORAL RESPONSE TO INFECTION

• Antibody production: Upon infection, B cells produce specific antibodies that target the invading pathogen. These antibodies can neutralize pathogens and mark them for destruction by other components of the immune system. 
• Specificity: The antibodies produced in the humoral response are specific to a  particular antigen, such as a viral protein or bacterial toxin. This specificity allows the immune system to target the specific pathogen causing the infection. 
• Diversity: The immune system has the ability to produce a large number of different antibodies, allowing it to recognize and respond to a wide range of pathogens. 
• Memory: The humoral response also results in the creation of long-lived memory B  cells and plasma cells that can rapidly respond to subsequent infections with the same pathogen.

Time course: The kinetics of the humoral response involves a lag phase after initial exposure to the pathogen, during which time the B cells are activated and begin to produce antibodies. This is followed by a rapid increase in antibody production,  reaching a peak after several days to weeks, and then a gradual decline. 

Booster response: Re-exposure to the same pathogen can lead to a rapid and stronger antibody response, known as a booster response, due to the presence of memory B cells.

ROLE OF CRP AND PROCALCITONIN 

C-reactive protein (CRP) and procalcitonin (PCT) are inflammatory biomarkers commonly used in the diagnosis of sepsis. CRP is an acute-phase protein that is synthesized in the liver under the stimulation of  IL-6 during infections or other inflammatory conditions.  

PCT is the precursor protein of calcitonin, which is normally synthesized by the C-cells of the thyroid gland, but in response to bacterial infections, multiple cell types throughout the body produce PCT. PCT has been found to be superior to CRP both in terms of sensitivity (77% vs  75%)and specificity (79% vs 67%) in the differentiation of bacterial septicaemia from noninfectious systemic inflammatory response syndrome.  

PCT levels are raised much earlier during an infectious process in comparison with  CRP (4–12 hours vs 24–38 hours), which facilitates earlier diagnosis. PCT has a plasma elimination half-life of 24–35 hours (vs 48 hours for CRP), which makes daily measurement of the levels clinically significant. A 30–50% daily drop in circulating PCT levels indicates that the infection is well controlled.

TESTING METHODS 

1.  Agglutination.  
2. Precipitation.  
3. Complement fixation Test (CFT).  
4. Neutralization.  
5. Immunofluorescence Test (IF).  
6. Enzyme-linked immunosorbent assay (ELISA).  
7. Radioimmunoassay (RIA).  
8. ImmunochromatographY (ICT).  
9. Immuno-blotting assays.

1. AGGLUTINATION 

This is the visible clumping of particulate (insoluble) Ag with its specific Ab forming a visible lattice. The Ab is the divalent agglutinating one either IgG or IgM type.  ▪The reaction can be either on slide or tube agglutination. It can be direct or via the use of carrier (Passive or indirect) e.g. Latex, RBCs  (hemagglutination), or Staph protein A (Coagglutination).  Indirect is more easily visible, and the reaction can be qualitatively or quantitatively expressed.

EXAMPLES 

• Direct slide detection of bacterial or viral antigens in a lesion or culture.  
• Direct tube agglutination (Classical Widal test) for diagnosis of typhoid fever  (replaced now by Latex)  
• Indirect Latex tests are commonly used in most microbiology laboratories nowadays.  
• Indirect passive hemagglutination tests e.g. Treponema Pallidum Hemagglutination  (TPHA) for diagnosis of syphilis 
• Brucella slide or tube agglutination test

Advantages of Agglutination:  

• Widely used tests for diagnosis and screening.  
• Easy and simple.  
• Very cheap  
• No instrument is required  
• somewhat sensitive 

Disadvantages of agglutination:  

• low specificity (have false positive results) need confirmatory tests 
• low sensitivity (have false negative results).  
• Results affected by vaccine-induced Ab.  
• Subjective errors in reading. 
• Agglutinating Ab appears late in the infections.

2. PRECIPITATION 

It the reaction between soluble Ag and its specific Ab,  in solution or in gel.  The Ab is incorporated into the gel and the Ag to be tested is the one allowed to  diffuse in the gel. Diameter of precipitation is proportional to the Ag concentration  directly.

Examples of Precipitation in solution include the Ring test for detection of infectious Ags  in CSF and  the slide test for detection of  pneumococcal capsule. Precipitation in gel is used for the quantification of Immunoglobulins in patient serum  (less common) in form of single, double radial immuno-diffusion or  immuno-electrophoresis.

3.COMPLEMENT FIXATION TEST (CFT)

This test is based on the capacity of complement to bind to antigen–antibody complexes.  When the complexes are present on the surface of red blood cells, complement  causes their lysis which can be visualized by a suitable experimental setup. Complement and the Ag used in the test are added in the lab to test serum after serum  heating to destroy the internal complement proteins and incubated in Wassermann  tubes.  Then, an RBCs and Anti-RBCs are added as indicator system to be destroyed by the  complement if there is no Ab in the serum. If there is Ab the complement will be fixed  in the first reaction so, no hemolysis will occur to certain dilution after which it will  occur.

Application of CFTs: 

• Bacterial: Syphilis and chronic gonorrhea. 
• Viral: Influenza. 
• Systemic fungal infections: Systemic candidiasis.

Advantages of CFT:  

• The CF Ab do not persist long (recent infections). 
• Stable antigen used in the test for years (>10 ys).  
• High specificity  
• Quantitative results.  

Disadvantages of CFT:  

• Low sensitivity (false negative)  
• Time consuming.  
• Not standardized  
• Needs high technical skills  
• Sample contamination giving anticomplentary results 
• Still false positive results occur. 

4. NEUTRALIZATION 

It is an Ag-Ab reaction in which the Ab neutralizes the effect of the Ag (bacterial  toxin or virus).
Example of neutralization test in vitro is the Anti- Streptolysin O titre (ASOT) for the  diagnosis of post- Streptococal Complications e.g. Rheumatic fever.

5. ENZYME LINKED  IMMUNOSORBANT ASSAY [ELISA]

• Monoclonal antibody (mAb) or recombinant Ag is fixed to a surface of a microtiter  plate wells.  
• The test sample (with unknown Ag or Ab) is applied and incubated to react then  washed.  
• The bound material is detected by a secondary, enzymatically labeled mAb  (Conjugate) which is incubated and then washed to remove unbound.  
• Then a substrate for the enzyme is added later on and the colored reaction is  stopped by acid. The color reaction is directly proportionate to the amount of the  unknown (Ag or Ab) present

ELISA Formats ▪Direct ELISA ▪Indirect ELISA ▪Sandwich ELISA

ADVANTAGES OF ELISA 

• Simplicity 
• Rapid 
•  Sensitivity- Detection levels of 0.01 to 1 μg/mL ideal for most diagnostic purposes.  
• Reagents Commercially available: with great flexibility and open to work in different  Equipment. 
• Cost: (a) Startup costs are low. (b) Reagent costs are low.
• Acceptability: Fully standardized ELISAs in many fields are now accepted as “gold standard”  assays.
• Safety: Safe non-mutagenic reagents are available. Disposal of waste has no problem  (unlike radioactive)
• Quantification of data allows easier standardization

Main drawbacks of ELISA is lacking the absolute specificity, so, still there are some  false-positive results to be confirmed by molecular testing.

6. IMMUNOFLUORESCENCE ASSAY  (IFA) 

It is Ag-Ab reaction where the Ab is labeled with fluorescent dye. The fluorescent dye  is either Fluorescin or Rhodamine linked to isothiocyanate (ITC).  The reaction is seen by the means of a special fluorescent microscope.  The IFA reaction can be direct to detect specific Ag by the means of labeling specific  Ab or indirect by using unlabeled Ab and fluorescently labeled Anti-Ab which is  better technique to only label one Anti-Ab and not all specific Abs to plenty of  Antigens needed to be detected. I IFA has the advantages of detecting Ags in tissues.

The most common example for IFA is Treponema Pallidum Immunofluorescent Antibody  test (TP-IFA) and infections caused by Chlamydia species .  

Advantages of IFA:  

• Fixed slides can persist for long time  
• High sensitivity.  

Disadvantages of IFA:  

• Subjective errors in reading.  
• Expensive equipment.

7. IMMUNOCHROMATOGRAPHY  (ICT)

• Immunochromatographic assays, termed also (Rapid Test), (Point-of-care test, POCT) or(lateral- flow dipstick) immunoassays.  
• Based upon Sandwich ELISA assay and run on chromatographic papers or Nitrocellulose  membrane by the capillary action.  
• The test strips or cartridge contains two type of fixed monoclonal antibodies one at the test  (T) zone which is specific to the Ag or Ab of interest (to be tested for), and the other Ab at  the control (C) zone which is anti-antibody to the human IgG for validation of reaction  components of the test strip or cartridge. 
• The 3rd type of mAb is the colloidal gold-labeled (Conjugate) free moving and infiltrating in  the strip or cartridge pad and specific to the Ag or Ab of interest (Tested for) and ready to  react with the Ag or Ab of interest (if present in sample) and move across the other side of  the strip
• If the test is positive the Ag or Ab will be trapped giving colored band at test (T)  zone and the human (e.g.) IgG will react with the Anti-antibody in the control (C)  areas giving colored band. •On the other hand, if the sample is negative for the Ag  or Ab of interest only colored band will appear at the C zone due to the presence of  human immuno- globulins indicating the validity of the test. 

If no colored band seen at the control (C), the test is invalid even if there is a band at  the Test zone 

Applications of Rapid tests in clinical diagnosis  

Wide range of usage of the rapid ICT tests including HIV, Hepatitis, malaria,  influenza, Pregnancy, filaria, Leishmannia, Trichomoniasis, Chagas disease ,  Entamoeba histolytica/dispar , Cryptosporidium and Giardia etc

Advantages:  

• Easy to use immediate and fast results (10-20 minutes) so used in screening. 
• Can be used at home or clinic.  
• Inexpensive  
• Some tests are FDA approved  
• Done upon whole blood or saliva or urine.  
• No equipment, refrigeration, No electricity, 
• No multiple timing steps required.  
• Built in controls 

Disadvantages of rapid tests.  

• oPositive late after infection; after seroconversion.  
• oNeed confirmation for reactive tests in some cases.  
• oSubjective variability in result reading.  
• oQualitative and not quantitative.  
• oLess sensitive than ELISA

8. IMMUNO-BLOTTING ASSAYS 

• Western blotting (WB) is analysis and detection of proteins was first developed in  1979.  
• The method is based on building an antibody: protein complex via specific binding of  antibodies to immobilized proteins on a membrane and detecting the bound antibody  with one of several detection methods.  
• WB is based on the principles of immunochromato- graphy where denaturated  proteins are separated into poly acrylamide gel according to the isoelectric point  and molecular weight by electrophoresis.  
• Then these separated proteins are transferred (blotted) on a nitrocellulose or  Polyvinylidene fluoride (PVDF) membrane.
• Specific labelled antibodies are allowed to react to these proteins fixed on the nitrocellulose or PVDF membrane (Ag-Ab reaction).  
• This Ag-Ab reaction then visualized according to the method of labelling of the antibody mainly staining and photography.  
• The photographed films can be kept for a long time.  

▪Several procedures and reagents are needed for sample preparation, gel electrophoresis, transfer, antibody probing, detection, imaging and analysis. 

APPLICATION OF AG-AB  REACTIONS (SEROLOGY):  

• Diagnosis of many infectious diseases (direct antigen detection or measuring Immunity to infection) especially for non-culturable or delayed-growing microorganisms.  
• Estimation of the Severity or stage of diseases.  
• Determination of the Response to Treatment.  
• Epidemiological studies.  
• Diagnosis of congenital infections.  
• Screening donation of blood and tissues.  
• Non-infectious diagnostic applications (Tumors, Autoimmune diseases, endocrinology

EXAMPLES OF SELECTED INFECTIOUS  ORGANISMS

Bacteria

A) GROUP A STREPTOCOCCUS (GAS) 

• Anti-Streptolysin O (ASO) test: The ASO test measures the level of antibodies against streptolysin O, a toxic enzyme produced by GAS. Elevated levels of ASO  indicate a recent or ongoing GAS infection. 
• Anti-Deoxyribonuclease B (Anti-DNase B) test: The Anti-DNase B test measures the level of antibodies against deoxyribonuclease B, another toxin produced by GAS.  Elevated levels of Anti-DNase B also indicate a recent or ongoing GAS infection. 
• Anti-Streptococcal Antibody Panel: This is a comprehensive test that measures the levels of multiple antibodies against GAS, including ASO and Anti-DNase B. The  Anti-Streptococcal Antibody Panel is useful for diagnosing and monitoring streptococcal infections and for differentiating between different types of streptococcal infections.

ADVANTAGES AND LIMITATIONS

• Rapid antigen detection test (RADT)- used to detect GAS antigens in a patient’s throat or skin swab. 
• ▪Highly sensitive for detecting GAS in the early stages of an infection. 
•  ▪Other advantages include ease of performance and quick TAT. 
• ▪Disadvantages include some false positive and negative results

B) SYPHILIS 

several serologic tests used to diagnose syphilis, include: 

• ▪Rapid Plasma Reagin (RPR) Test: This is a non-treponemal test that measures the presence  of antibodies against cardiolipin, a component of the host’s own cells. The RPR test is rapid,  inexpensive, and widely available, making it a commonly used screening test for syphilis. 
• ▪Venereal Disease Research Laboratory (VDRL) Test: This is a non-treponemal test that  measures the presence of antibodies against cardiolipin, similar to the RPR test. The VDRL  test is widely used in clinical settings, and it is considered to be highly specific for syphilis. 
• ▪Fluorescent Treponemal Antibody-Absorption (FTA-ABS) Test: This is a treponemal test  that specifically detects antibodies against Treponema pallidum. The FTA-ABS test is highly  specific for syphilis, and it is considered the gold standard for confirming a syphilis  diagnosis.

Other treponal tests include;  

• microhemagglutination assay T pallidum (MHA-TP)  
• T pallidum hemagglutination (TPHA) 
• T pallidum particle agglutination (TPPA) tests. 
• Treponemal enzyme immunoassay (EIA) for immunoglobulin G (IgG) and  immunoglobulin M (IgM) may be performed.

IMMUNOLOGIC VIRAL DIAGNOSTIC  METHODS 

1.Direct Detection of Viral Antigens in Clinical Specimens 

▪Methods of antigen detection include fluorescent antibody (FA) staining,  immunoperoxidase staining, and EIA. 

▪FA staining is the most widely used in diagnostic virology 

▪The method was widely adopted by clinical laboratories during the 1980s,  particularly for detection of respiratory viruses.  

▪Antigen detection methods are particularly useful for viruses that grow slowly or are  labile, making recovery in culture difficult 

▪Viruses diagnosed include RSV, influenza and parainfluenza viruses, and adenovirus  in respiratory specimens; HSV and VZV in cutaneous specimens; rotavirus in stool  specimens; and CMV and hepatitis B virus (surface antigen) in blood specimens. 

2. DETECTION OF ANTIBODY  RESPONSE TO VIRUS INFECTIONS

• ▪IgM detection is primarily useful when viral detection methods are not readily available or  are too slow to give clinically useful results
• ▪IgM antibodies can usually be detected within 5 to 7 days of clinical symptoms, but may  require up to 2 weeks.
• ▪For some virus infections, the clinical symptoms prompting medical attention are immune  mediated; therefore, IgM is usually detectable when the patient first presents.  

▪Examples include hepatitis A virus (HAV), HBV, parvovirus B19, and Epstein-Barr virus (EBV).  

▪Antibodies to several distinct viral antigens provide useful information regarding the stage of  HBV and EBV infections.

LIMITATIONS OF IgM TESTING 

• ▪Variability in test methods 
• ▪Weak or undetectable antibody responses in immunocompromised patients or  neonates 
• ▪Heterologous IgM responses 
• ▪Interference by IgM-class rheumatoid factor (RF) 
• ▪Competition between IgG and IgM antibodies leading to false positives and false  negatives 
• ▪Failure to detect IgM due to timing of sample collection 
• ▪Persistence of IgM in some chronic viral infections 
• ▪Frequent absence of IgM in reinfections or reactivations.

IMMUNODIAGNOSIS OF PARASITIC  INFECTIONS

• ▪Definitive diagnosis of parasitic infections is made by identification of parasites in  properly collected specimens or affected tissues. 
• ▪Identification is not generally possible in when parasites are located in deep tissue  sites e.g toxoplasmosis or where invasive techniques with some risk to the patient are  necessary to obtain material e.g. cysticercosis  
• ▪Most parasitic infections induce a vigorous humoral immune response, so the presence  of parasite-specific antibodies can be a valuable indicator of infection, especially if  the patient has no prior history of exposure.
• ▪Abs may persist for as little as 6 months after infection or treatment or for many  years 
• ▪Humoral immune response usually precedes clinical manifestations for most parasitic  infections. 
• ▪ Detection of IgA and IgE alone is not informative, requires IgG detection that are  specific to parasite or its antigen component. Except in Toxoplasma-specific IgM and  IgA as indicators of acute neonatal toxoplasmosis

SELECTED PARASITIC INFECTION

a) African trypanosomiasis – 

• Detection of specific antibodies is the best method for screening, while diagnostic confirmation and  staging require examination of csf.  
• Card agglutination test for trypanosoma (CATT) is the most reliable test available for screening.   CATT utilizes whole ,fixed, Coomassie blue-stained trypanosomes that react with serum IgG or IgM  Abs to form visible precipitate/agglutination. 

b) Cysticercosis (larva Tinea solium)  

• Enzyme linked immunoelectrotransfer blot (EITB) are the antibody detection test formats most  frequently used for the diagnosis of NCC (Neurocysticercosis). 
• utilizes partially purified T. solium antigens.  
• Detects antibodies to one or more of seven lentil lectin-bound glycoprotein (LLGP) present in an extract  of T. solium cysts.

c)Amebiasis 

• Entamoeba histolytica is associated with both intestinal and extraintestinal infections.  Ab detection is the most useful in patients with extraintestinal disease, i.e. amebic liver abscess  Antigen used is crude soluble extracts of axenically cultured organisms.  
• Enzyme immunoassay and indirect hemagglutination assay are mostly used.  Immunodetection of E. histolytica antigens in fecal specimens may be used to distinguish the  morphologically identical pathogenic E. histolytica and non-pathogenic E. dispar. The assay detect the galactose inhibitable adherence proteins, which is necessary for pathogenesis.

d) Malaria Caused by Plasmodium spp.  

• Rapid diagnostic technique uses histidine rich protein-2 of Plasmodium falciparium and lactate  dehydrogenase, a pan malarial parasite antigens.  
• Antibody against these antigen is detected in serum sample. 

e)Leishmaniasis Caused by Leishmania donovanii.  

• Immunodiagnosis technique detect antibody against cultured epimastigotes of various Leishmania spp.,  or crude solubilized epimastigotes.  
• Rapid immunochromatographic card test use recombinant proteins k39 or rK28.
  

IMMUNODIAGNOSIS OF FUNGI 

• Invasive fungal infections(IFI) present a great challenge nowadays, esp. in population  with immunosuppression.  
• ▪ Cultures, though the gold standard of diagnosis, is time consuming and has low  sensitivity 
• ▪Detection of specific host immune responses to fungal antigens using immunologic  reagents is an alternative. 
• ▪Targets for serological tests are – Antigen – Antibody – Metabolites  
• ▪ Decision of fungal serologic test is based on – clinical presentation – exposure history  – risk factor for infection

FUNGAL PATHOGENS OF MEDICAL IMPORTANCE OPPORTUNISTIC  

• Candida species  
• Aspergillus species  
• Cryptococcus species  
• Pneumocystis jiroveci  

TRUE PATHOGENS  

• Coccidioides immitis  
• Paracoccidioides brasiliensis  
• Histoplasma capsulatum  
• Blastomyces dermatitidis

1.CRYPTOCOCCOSIS 

• Capsulated yeasts  
• ▪Polysaccharide antigen ((glucuronoxylomannan) Serum>CSF>Urine 
•  ▪Detection of the capsular antigen is confirmative of Cryptococcal infection  ▪Qualitative & semi-quantitative test  
• ▪Sample – serum , CSF, bronchoalveolar lavage  
• ▪Significant titre ≥1:8  
• ▪Sensitivity – 90.9% Specificity – 95%  
• ▪False positives – Rheumatoid factor, Trichosporon asahii, Stomatococcus  ▪ False negative – Prozone phenomenon

2.INVASIVE CANDIDIASIS 

▪Detection of Heat labile glycoprotein (HLP)  

▪Detection of Mannan antigen (Pastorex Candida Antigen)  ▪Detection of anti –Mannan antibody (Pastorex Candida Antibody)  ▪False positives

3. Blastomycosis –Blastomyces dermatitidis 

Sample – serum, CSF  

Qualitative assay  

Detection of precipitating antibodies to purified A antigen  Specificity ≥90% • Sensitivity – low in early infection or localised disease  Long turnaround time

4. Histoplasmosis-Histoplasma capsulatum  

Antigens  

M protein –abundant in mycelial form 

H protein – indicative of active infection  

Antibodies 

 Anti M Ab – develop soon after infection – lasts upto 3 yrs after resolution  Anti H Ab – alone /with Anti-M Ab indicates active/recent infection

IMMUNOLOGIC DIAGNOSIS OF  TUBERCULOSIS 

▪Include serological assays against various mycobacterial antigens. 

▪Serological assays have a high negative predictive value, making them potentially useful as screening test to rule out active TB. 

▪In HIV-positive individuals, low sensitivity and low negative predictive value compromise the accuracy of the seroassays. 

▪In populations where the prevalence of latent TB infection is high, the relatively low  positive predictive value of the tests reduces their specificity for active TB

SPECIFIC SEROLOGICAL TESTS FOR  DIAGNOSING TUBERCULOSIS 

Antigen 5 (38 kDa antigen) 

• ▪ Antigen 5 is a protein antigen that is found in Mycobacterium tuberculosis 
• ▪The use of antigen 5, also known as the 38 kDa antigen, in a microtiter plate ELISA  has been evaluated in a number of countries with reasonable sensitivity and high specificity. 
• ▪Generally, the antibody titer correlated with the extent of pulmonary disease

A60 antigen 

• ▪A60 antigen, a thermostable component of PPD, has also been used in the serodiagnosis of TB. 
• ▪It is not specific for mycobacteria because it is also present in Nocardia and  Corynebacterium species. 
• ▪In a study of 560 Chinese patients with pulmonary and extrapulmonary TB and over  700 controls, measurement of IgM appeared to be sensitive (80%) for active primary  TB and specific (100%) for latent TB

30 kDa antigen 

• ▪Protein antigen that is found in Mycobacterium tuberculosis 
• ▪The specificity for dot EIA and plate ELISA were 92% and 97%, respectively; the  sensitivity rate for hospitalized patients with TB were 69% and 78%, respectively 

Antigen p90 

• ▪Arikan et al.37 showed that in 51 patients with active TB, the sensitivity of anti-Kp90  IgA in sera or body fluids was 82% and in 71 control patients, the specificity was  90%

Cord factor 

• ▪Measures IgG antibody response to M. tuberculosis cord factor (trehalose-6,6’  -dimycolate). 
• ▪Overall sensitivity of patients with any bacterial or clinical diagnosis of a  mycobacterial infection was 83% and the specificity was 100%. 
• ▪Antibody titers declined to normal levels with antituberculous chemotherapy.

Lipoarabinomannan 

• LAM is comprised of a linear series of ringed mannose sugar residues, with occasional branches of single mannose residues. 
• It is known to have a number of immunomodulatory effects. 
• The detection of anti-LAM antibodies can be used in the serological diagnosis of active TB. 
• The specificity of the test is excellent, ranging from 84–100%. 
• In patients with active TB, the sensitivity of anti-LAM IgG is 85–93%

Conclusion

Immunodiagnosis continues to play a critical role in the diagnosis of bacterial, viral, fungal and parasitic infections. They are inexpensive, widely available and most do not require high technical expertise. Serological tests are especially useful as screening tools. Their main drawback is lower sensitivity and specificity compared to other diagnostic techniques.