Genes for the human alpha 5(IV) and alpha 6(IV) collagen chains have a unique arrangement in that they are colocalized on chromosome Xq22 in a head-to-head fashion and appear to share a common bidirectional promoter. In addition we reported a novel observation that the COL4A6 gene is transcribed from two alternative promoters in a tissue-specific manner (Sugimoto, M., T. Oohashi, and Y. Ninomiya. 1994. Proc. Natl. Acad. Sci. USA. 91:11679-11683). To know whether the translation products of both genes are colocalized in various tissues, we raised alpha 5(IV) and alpha 6(IV) chain-specific rat monoclonal antibodies against synthetic peptides reflecting sequences near the carboxy terminus of each noncollagenous (NC)1 domain. By Western blotting alpha 6(IV) chain-specific antibody recognized 27-kD monomers and associated dimers of the human type IV collagen NC1 domain, which is the first demonstration of the presence in tissues of the alpha 6(IV) polypeptide as predicted from its cDNA sequence. Immunofluorescence studies using anti-alpha 6(IV) antibody demonstrated that in human adult kidney the alpha 6(IV) chain was never detected in the glomerular basement membrane, whereas the basement membranes of the Bowman's capsules and distal tubules were positive. The staining pattern of the glomerular basement membrane was quite different from that obtained with the anti-alpha 5(IV) peptide antibody. The alpha 5(IV) and alpha 6(IV) chains were colocalized in the basement membrane in the skin, smooth muscle cells, and adipocytes; however, little if any reaction was seen in basement membranes of cardiac muscles and hepatic sinusoidal endothelial cells. Thus, both genes are expressed in a tissue-specific manner, perhaps due to the unique function of the bidirectional promoter for both genes, which is presumably different from that for COL4A1 and COL4A2.

A group of rat monoclonal antibodies recognizing the six different alpha chains of human type IV collagen have been established by our novel method. The method is designated the rat lymph node method in which enlarged medial iliac lymph nodes of a rat injected with an antigen emulsion via hind footpads are used as a source of B cells for cell fusion to produce hybridomas. The immunogens used were synthetic peptides having non-consensus amino acid sequences near the carboxyl termini of type IV collagen alpha chains. Hybridomas were screened both by ELISA with synthetic peptides and by indirect immunofluorescence with cryostat sections of human kidneys. Because the epitopes of all antibodies were determined by multipin-peptide scanning, they were confirmed to be isoform-specific. They are useful for identification of alpha chains of type IV collagen at the protein level in normal and abnormal conditions. The combined use of synthetic peptides as immunogens, the rat lymph node method as making monoclonal antibodies, and the multipin-peptide scanning as epitope mapping is found to be a strong tool for identification of peptides and proteins whose amino acid sequences are known or have been deduced.

Key words: rat monoclonal antibody/medial iliac lymph node cells/rat-mouse hybridomas/cell fusion ABSTRACT.A novel method of preparing hybridomas producing rat monoclonal antibodies was established. The enlarged medial iliac lymph nodes from rats injected via hind footpads with an emulsion of antigen and Freund's adjuvant were used for cell fusion. Ovalbuminwas used as a representative antigen. The incidence of hybridomas producing antibody of interest with this method was about 10 times higher than that of hybridomas with the conventional method using mouse or rat spleen cells. The average percentages of hybridomas producing IgGl, IgG2a, IgG2b and IgG2c were 37.1%, 47.0%, 15.9% and 0.0%, respectively. A single injection with antigen was sufficient for immunization in this method.In 1975, Kohler and Milstein first reported the cell fusion technique that made it possible to generate hybrid cell lines producing monoclonal antibodies (7). Since then, monoclonal antibodies have been used increasingly so as to be applied in almost all areas of life science. Reagents and myelomas for cell fusion have been improved (2, 3, 4, 5,16), and hybridoma technology is now firmly established. It is still difficult, however, to obtain desired monoclonal antibodies because of low incidence of positive hybridomas (i.e., hybridomas producing antibody of interest) when spleen cells are used as a source of B cells. In the past, we have established several experimental models of anti-glomerular basement membrane antibody-induced glomerulonephritis in rats (ll, 12, 13, 14). Wefound in the early stage of our research that footpad injection as well as intraperitoneal injection of the nephritogenic antigen could induce severe nephritis in rats and that the rats showed hypertrophic medial iliac lymph nodes. We speculated that the medial iliac lymph nodes could be applied as a reliable source of B cells for producing hybridomas yielding monoclonal antibodies.In this paper, we propose a novel method of producing monoclonal antibody comparing it with conventional methods. Our method is termed the rat lymph node method which consists of hind footpad injection of rat * To whomcorrespondence should be addressed. Abbreviations: BSA, bovine serum albumin; ELISA, enzymelinked immunosorbent assay; GBM, glomerular basement membrane.and subsequent cell fusion with the enlarged lymph nodes. The conventional methods are termed mouse and rat spleen methods which consist of intraperitoneal immunization of an animal and subsequent cell fusion with the spleen. Ovalbuminwas used as a representative antigen.The first half of this paper deals with comparison of the novel method and conventional methods. Wefound that the rat lymph node method was about 10 times moreeffective as the mouseand rat spleen methods.The latter half of this paper describes factors that may affect the incidence of positive hybridomas in the rat lymph node method. The effect of booster injection, the timing of the cell fusion, and the use of the inguinal lymph nodes as a fusion partner ...

Temporal and spatial expression of alpha1 (IV), alpha2 (IV), alpha3 (IV), alpha4 (IV), alpha5 (IV), and alpha6 (IV) collagen chains was studied during the formation of the basal lamina in an "in vitro" skin model. A sequential study was performed at 7-d and 14-d cultures (lamina densa absent) and at 28-, 36-, and 56-d cultures (lamina densa present). Expression of beta1, beta4, alpha1, alpha2, alpha3, alpha5, alpha6 integrin subunits and co-localization with collagen IV was studied by regular and laser confocal indirect immunofluorescence microscopy. mRNA expression of alpha2 (IV) and alpha6 (IV) chains was estimated by northern blots. The earliest expression of alpha1 (IV) and alpha2 (IV) collagen chains was noted in 7-d cultures restricted to basal keratinocytes. At 14-d cultures, alpha1 (IV) and alpha2 (IV) chains were noted in basal keratinocytes and as a broad band (10 microm) in the adjacent dermis. At this stage 80% of the alpha2 (IV) mRNA was expressed in the dermis and 20% in the epidermis. At 28-, 36-, and 56-d cultures the alpha1 (IV) and alpha2 (IV) chains were present in a linear distribution at the epidermo-dermal junction and in the upper dermis. The alpha6 (IV) collagen chains were expressed much later at 36-d cultures and the alpha5 (IV) at 56 d, both mostly in a linear distribution but also in the adjacent dermis. Alpha6 (IV) mRNA was demonstrated in the dermis of 36-d cultures. There was co-localization of collagen IV and beta1 integrin subunits in 14-d cultures at the matrix site of keratinocytes. Functional perturbation studies with AIIB2 monoclonal antibody (anti-beta1 subunits) and competitive inhibition with a collagen cyanogen bromide digestion derived fragment (CB3[IV]) that contains the collagen IV ligand for alpha1beta1, alpha2beta1 integrins, altered the pattern of collagen IV deposition.

We first completed the primary structure of the mouse alpha5(IV) and alpha6(IV) chains, from which synthetic peptides were produced and a chain-specific monoclonal antibodies were raised. Expression of collagen IV genes in various basement membranes underlying specific organ epithelia was analyzed by immunohistochemical staining using these monoclonal antibodies and other antibodies from human and bovine sequences. It was possible to predict the presence of the three collagen IV molecules: [alpha1(IV)](2) alpha2(IV), alpha3(IV)alpha4(IV)alpha5(IV), and [alpha5(IV)](2)alpha6(IV). In skin basement membrane two of the three forms, [alpha1(IV)](2)alpha2(IV) and [alpha5(IV)](2)alpha6(IV), were detected. The alpha3(IV)alpha4(IV)alpha5(IV) molecule was observed as the major form in glomerulus, alveolus, and choroid plexus, where basement membranes function as filtering units. The molecular form [alpha5(IV)](2)alpha6(IV) was present in basement membranes in tubular organs such as the epididymis, where the tubes need to expand in diameter. Thus, the distribution of the basement membranes with different molecular composition is consistent with tissue-specific function.

The direct double immunofluorescence staining of kidney and skin cryostat sections with the fluorochrome-conjugated antibodies is useful, reliable, and convenient for diagnosis of Alport's syndrome.

Nephritogenicity (anti-GBM-nephritis-inducing activity) and alpha-chain composition of globular-do-main (NCI) fractions of type IV collagen from bovine renal, pulmonary, and placental basement membranes (BMs) was examined by injecting these fractions with adjuvant into WKY/NCrj rats and by Western blotting using epitope-defined monoclonal antibodies to the six different alpha chains of type IV collagen. A purified nephritogenic fraction from renal BM contained alpha 1-alpha 6(IV)NCI, whereas a non-nephritogenic fraction contained only alpha 1-alpha 2(IV)NCI. Renal and pulmonary NCI had strong nephritogenic activity: placental NCI had weak activity. The renal and pulmonary fractions contained alpha 1-alpha 6(IV)NCI, and the placental fraction had a large amount of alpha 1-alpha 2(IV)NCI and a very small amount of alpha 3-alpha 6(IV)NCI. Immunohistochemical study of bovine renal BM with the monoclonal antibodies revealed that bovine glomerular BM contained alpha 1-alpha 5(IV) chains, but not the alpha 6(IV) chain. The absence of alpha 6(IV) chain in glomerular BM in bovine and in humans indicates that alpha 6(IV) chain is not a target antigen of anti-GBM nephritis. Nephritogenicity is apparently a property of alpha 3-alpha 5(IV)NCI.

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