CORRELATION OF AUTOANTIBODIES PRESENCE DETECTED BY IFA-anti- dsDNA, IFA-AMA AND IMMUNOBLOTTING WITH CORRESPONDING DATA IN CLINICAL MANAGEMENT OF AUTOIMMUNE DISEASES

Diagnosis and management of patients with SLE (Systemic Lupus Eritematosus), autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC), involves specifi c diagnostic tests, such as IFA-AMA, IFA antidsDNA and immunoblotting for the detection of autoantibodies for specifi c autoantigens (mitochondria, dsDNA, M, LKM-, LC-, SLA/LP). We established specifi c correlation between the detected autoantibodies and corresponding clinical fi ndings. Th e total of  serum specimens were probed with IFA-anti-dsDNA,  of which tested positive. We also performed dilution analysis to the end point for all the positive specimens. Numerous specimens were tested by IFA, AMA and immunoblotting.


Introduction
Systemic Lupus eritematosus (SLE) is one of characteristic autoimmune diseases in which numerous autoantibodies are produced.Glomerulonephritis and arthritis are often among other clinical manifestations.Immune complexes include autoantibodies and DNA or nucleoprotein antigens.Th e most frequent type of autoantibodies in SLE patients, are anti-dsDNA.Also, there are autoantibodies against ribonucleoproteins, histones and nucleolus.Autoantibodies against erytrocytes and platelets are characteristic for hemolytic anemia and thrombocytopenia.Autoantibodies against native double stranded (ds) DNA are specifi c for SLE and thus, Crithidia lucillae anti-dsDNA indirect immunofl uorescence test (IFA anti-ds-DNA) is the principal diagnostic test.().Hemofl agelate Crithidia lucillae possesses a modifi ed mitochondrion called kinetoplast, which naturally contains numerous molecules of double stranded DNA (ds-DNA) that are not associated with histones.Kinetoplast is a spherical organelle, smaller than nucleus, located near nucleus and basal body.Th e IFA anti-dsDNA diagnostic test is highly specific and moderately sensitive in the management of diseases such as SLE which is predominant in women (incidence : , age between - years, male to female ratio :).Th e main clinical features include: rashes, arthritis, glomerulonephritis, hemolytic anemia, thrombocytopenia (, ).In the suspect cases of liver diseases or autoimmune hepatitis, early and fast detection of the following human autoantibodies of the class IgG, against four different specific antigens, is very important: -M (pyruvate dehidrogenase complex) -LKM- (liver -kidney microsomescytochrome P II-D) -LC - (cytosolic liver antigen type -) -SLA/LP (soluble liver antigen/ liver -pancreas antigen).
Diagnostic Western blot method offers the possibilities of fast, highly sensitive, highly specifi c and simple procedure for the detection of autoantibodies against the above mentioned specific antigens, in either serum or plasma, and gives the chance for timely and successful treatment of patients.Specially designed immmunoblot strips contain parallel lines of antigens purified by affinity chromatography.Specific IgG, IgA and IgM antibodies bind to corresponding antigen site.Th e detection is based on enzyme-labeled conjugate promoting a color reaction.No cross-reactions with other autoantibodies were found ().
During one year, we tested the total of  clinical blood specimens for presence of specific autoantibodies against M, LKM-, LC- and SLA/ LP antigens by Western blot method (Euroimmun).We also analyzed the total of  serum specimens by IFA anti-dsDNA and  specimens by IFA-AMA.Th e tests were performed according to the obtained recommendations of clinicians at University of Sarajevo Clinics Centre.

Material and Methods
Serum specimens were collected from patients at diff erent Clinics at University of Sarajevo Clinics Centre.

Immunofl uorescence test
We performed IFA anti dsDNA test using BioSystems anti nDNA antibodies test designed for this purpose.

Results
Serum

Discussion
Primary biliary cirrhosis (PBC) is a chronic autoimmune disease characterized by the destruction of small intraliver bile ducts.Determination of antimitochondrial antibodies (AMA) is of particular signifi cance for proper diagnosis and PBC management.It is also useful in other cases of liver diseases of unknown etiology.IFA is a gold standard in analyzing suspect cases of AIH, PBC, SLE, Sjogren's syndrome and rheumatoid arthritis (sensitivity  and specifi city ,).Also, another test with high sensitivity and specifi city ( and ) for the detection of specifi c PBC autoantigens is ELISA-M test.Molecular targets for these autoantibodies are members of -oxoacid dehydrogenase complex family of enzymes in the mitochondrial respiratory chain and as well as lipoyl binding domains (E).In clinical PBC management it is very important to detect these specifi c autoantigens.
In the suspect cases of liver diseases or autoimmune hepatitis, early and fast detection of following human autoantibodies of the class IgG against four different specific antigens, is very important: -M (pyruvate dehidrogenase complex) -LKM- (liver -kidney microsomes -cytohrome P II-D) -LC - (cytosolic liver antigen type -) -SLA/LP (soluble liver antigen/ liver -pancreas antigen).
Diagnostic Western blot method offers the possibilities of fast, highly sensitive, highly specifi c and simple procedure for the detection of autoantibodies against the above mentioned specific antigens, in either serum or plasma, and gives the chance for timely and successful treatment of patients.Specially designed immmunoblot strips contain parallel lines of antigens purifi ed by affi nity chromatography.Specifi c IgG, IgA and IgM antibodies bind to corresponding antigen site.Th e detection is based on enzyme-labeled conjugate promoting a color reaction.No cross-reactions with other autoantibodies were found (, , ).
In management of liver autoimmune diseases such as primary biliary cirrhosis (PBC) and autoimmune hepatitis (AIH), it is very important to exclude other causes e.g.viruses, alcohol and drugs.The main criteria for AIH diagnosis are: -histological fi ndings, -detection of autoantibodies (ANA, SMA, LKM, SLA/LP), -hipergammaglobulinaemia, -negative HB and HCV serology, -detection of HLA antigens B, DR or DR.
At least four of these criteria must be determined for appropriate diagnosis of AIH.If all criteria are present the diagnosis is defi nite.Final confi rmation is achieved through good response of patients to immunosupresive therapy.Circulating autoantibodies have great signifi cance for AIH diagnosis.Because of unclear correlation between the titre and activity or disease prognosis, the role of these antibodies in the AIH pathogenesis remain unclear.Th e autoimmune hepatitis is mainly specifi c for women with incidence of , cases per . individuals in Western Europe.Without corresponding timely diagnosis and treatment, AIH often progresses to liver cirrhosis.SLA/LP autoantibodies are highly specifi c for AIH and have not been described in viral hepatitis.Early, highly specific, sensitive and fast diagnosis provides the patient with a chance for normal life.Th ere are three subtypes of AIH: -subtype I (presence of ANA and SMA autoantibodies) -subtype II (autoantibodies against LKM- antigen) -subtype III (SLA/LP autoantibodies) Autoantibodies against LKM- can be found in - of patients with positive hepatitis C serology.For primary biliary liver cirrhosis autoantibodies against M are specific.Clinical overlap between patients with progressive systemic sclerosis and patients with primary biliary liver cirrhosis, in the case of M autantibodies finding is possible.Six proteins were identified as M antigens with following molecular mass: , , , ,  and  kDa (, , , , , ).
Our IFA-anti dsDNA, AMA, imunobloting analyses of serum specimens, as a part of AIH PBC, SLE diagnostic approach show significant correlation with final clinical diagnosis of these diseases.

Conclusion
During one year, we analyzed clinical blood specimens for the presence of specifi c autoantibodies against M, LKM-, LC- and SLA/LP antigens by Western blot method (Euroimmun).In combination with other analysis such as histological fi ndings, antinuclear antibodies (ANA), smooth muscle cells antibodies (SMA) and other testing, it is possible to perform fast and timely diagnosis of AIH and PBC.Th e results obtained after IFA-AMA and IFA-anti-dsDNA testing has improved diagnostics of AIH, PBC, SLE and RA.Among other diagnostic tools they are important parameters in early diagnostics of these diseases as well as a part of continuing process of monitoring the therapy and its eff ects.

List of
specimens were collected from patients at diff erent Clinics at University of Sarajevo Clinics Centre.Th e serum specimens were tested by IFA anti dsDNA, IFA-AMA and LKM immunobloting strips for the presence of specifi c autoantibodies: -autoantibodies against dsDNA -antimitochondrial autoantibodies (AMA) -against M-pyruvate-dehydrogenase complex -against LKM--liver-kidney microsomes -against Cytochrome pIID -against LC--cytosolic liver antigen type  -against SLA/LP -soluble liver antigen/liver pancreas antigen Th e results obtained by immunofl uorescent and immunoblotting analyses are summarized in Tables ,  and  and illustrated by corresponding fi gures (, ,  and ) and diagrams (, ,  and ).
SUBAŠIĆ ET AL.: CORRELATION OF AUTOANTIBODIES PRESENCE DETECTED BY IFAANTIDSDNA, IFAAMA AND IMMUNOBLOTTING WITH CORRESPONDING DATA IN CLINICAL MANAGEMENT OF AUTOIMMUNE DISEASES basal body or fl agellum illumination is not positive fi nding for anti-dsDNA ( , ,  ).
Before the commencement of test, the reagents and serum specimens are maintained at room temperature for at least half an hour.PBS buff er is diluted with distilled water to fi nal x concentration.Serum specimens are diluted with PBS buff er.One drop (μl) of diluted serum, specimens and controls, are added into slide wells.After the incubation and rinsing procedures, IgG FITC-EVANS is added and fi nally, several drops of mounting medium.Th e slides are examined by fl uorescence microscope ( nm excitation fi lter and  nm emission fi lter).In order to obtain the best results the slides are analyzed immediately.Positive serum specimens are titrated to the end point dilution defi ned as the highest dilution giving positive result.Finding is positive only in the case of kinetoplast fl uorescent illumination.Nuclei, ĐEMO human IgG), and fi nally for  minutes in , ml substrate solution.Incubation steps are performed at room temperature on rocking shaker.Th e immunoblot strips are washed x minutes with diluted wash buff er.Strips are than air-dried and the results evaluated based on signal intensity (negative, borderline, positive and strong positive).Only the strips with strong color reaction in the control band are considered valid(, , , , ).

TABLE 1
SUBAŠIĆ ET AL.: CORRELATION OF AUTOANTIBODIES PRESENCE DETECTED BY IFAANTIDSDNA, IFAAMA AND IMMUNOBLOTTING WITH CORRESPONDING DATA IN CLINICAL MANAGEMENT OF AUTOIMMUNE DISEASES . IFA testing results of serum specimens Six patients (age from  - years ) from Clinic for Gastroenterohepathology with positive AMA, were diagnosed with liver disease, as follows: Patient .Dg.Cirrhosis hepatis compensata; Hypertensio portalis Patient .Dg.Cirrhosis hepatis decompensata Patient .Dg.AIH; Cirrhosis hepatis micronodularis inc.Patient .Dg.Cirrhosis hepatis biliaris primaria susp.Patient .Dg.Hepatitis chronica B Patient .Dg.Cirrhosis hepatis decompensata According to our results, diagnosed diseases at corresponding Clinics were: SLE, RA AIH and PBC.TABLE 3. Immunoblot testTABLE 3. Th e origin of serum specimens analyzed for IFA and immunoblot -overview per Clinics ĐEMO ĐEMO SUBAŠIĆ ET AL.: CORRELATION OF AUTOANTIBODIES PRESENCE DETECTED BY IFAANTIDSDNA, IFAAMA AND IMMUNOBLOTTING WITH CORRESPONDING DATA IN CLINICAL MANAGEMENT OF AUTOIMMUNE DISEASES